Compositions and methods for reducing germ cell death

ABSTRACT

Described herein are methods of protecting germ cells in a subject from cell death induced by a chemotherapeutic agent by, in part, administering a composition comprising one or more humanin polypeptides to the subject. Also described herein are methods of inhibiting a reduction or decrease in fertility in a subject wherein the reduction or decrease in fertility is due to a chemotherapeutic agent.

RELATED PATENT APPLICATIONS

This patent application claims the benefit of U.S. Provisional PatentApplication No. 61/951,198 filed on Mar. 11, 2014, and U.S. ProvisionalPatent Application No. 61/951,174 filed on Mar. 11, 2014, each of whichapplications are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created May 22, 2015, isnamed LaBioMed0437579.ST25txt and is 25,003 bytes in size.

FIELD

The technology relates in part to compositions that comprise humaninand/or humanin analogs and uses thereof.

INTRODUCTION

Chemotherapy is a relatively effective drug treatment designed to killcancer cells in individuals with various forms of cancer. However, theadministration of a chemotherapeutic agent (e.g., type, dosing andfrequency of administration) must be weighed against certain adverseside effects of such a treatment. Chemotherapeutic agents sometimes killcancer cells as well as “normal” non-cancerous cells. Thus there is aneed for improving the efficacy of chemotherapeutic agents to morespecifically target and/or to more effectively kill cancer cells whilereducing the toxic affects of such agents on non-cancerous cells.

One problem associated with certain chemotherapeutic regimes ischemotherapy-induced sterility. Chemotherapy is often the first-line oftreatment in men for many cancers, including for example leukemia,lymphoma, testicular tumors, central nervous system tumors, andmelanomas. After cycles of combined cancer chemotherapy many young menhave long term cancer-free survival but are infertile becausechemotherapeutic drugs induced cell death of immature germ cellsincluding spermatogonial stem cells.

Cyclophosphamide (CP) is an example of a chemotherapeutic agent used inmen and in experimental animals. CP treatment causes germ cell damage inrodents and men but requires liver cytochrome P450 metabolism togenerate an active metabolite, 4-hydroxy-cyclophosphamide, whichcirculates to cancer cells and damages DNA leading to apoptosis(Sloderbach, et al., 2013). During in vivo treatment with CP, alldifferentiated germ cells of mice are eliminated and about half of theundifferentiated germ cells remain. Once treatment is stopped, about 64%of germ cells regenerate from these undifferentiated stem cells(Drumond, et al., 2011). Even though CP does not completely eliminatestem cell spermatogonia in the short term, stem cell spermatogonia lossmay not be completely reversible in cancer patients after multiplecycles of cancer chemotherapy. Some recovery has been reported inrodents (Delbes, et al., 2010) and men (Dohle, 2010; Meistrich, 2013;Trost & Brannigan, 2012). Currently in men, cryopreservation ofspermatozoa is a recommended method to preserve male fertility inpost-pubertal men and experimental cryopreservation of testicular tissuefor prepubertal boys. However, in some cases where a cancer ismetastatic and the patient is severely ill, spermatogenesis may beimpaired and the quality of a sample collected before treatment may bepoor and not suitable for use in assisted reproductive technologies(Choy & Brannigan, 2013; Loren, et al., 2013; Nangia, et al., 2013).

Compositions and methods disclosed herein can alter the effects ofcertain chemotherapeutic agents resulting in, for example, protection ofgerm cells from chemotherapy-induced death and/or enhancement ofchemotherapy-induced death of cancer cells.

SUMMARY

Provided herein, in some aspects, is a method of protecting germ cellsin a subject from cell death, wherein germ cell death is stimulated by achemotherapeutic agent, the method comprising administering to a subjecttreated with a chemotherapeutic agent an amount of humanin or a humaninanalog sufficient to protect germ cells in the subject from cell death.In some embodiments cell death is apoptotic cell death.

Provided herein, in some aspects, is method of inhibiting decreased orreduced fertility in a subject caused by treatment with achemotherapeutic agent, comprising administering to a subject treatedwith a chemotherapeutic agent an amount of humanin or a humanin analogsufficient to inhibit decreased or reduced fertility in the subjectcaused by treatment with a chemotherapeutic agent.

In certain aspects, a humanin or a humanin analog does not substantiallyreduce, decrease, suppress or inhibit efficacy or activity of thechemotherapeutic agent.

In some embodiments humanin comprises the sequence:MAPRGFSCLLLLTSEIDLPVKRRA. In some embodiments a humanin analog comprisesthe sequence: MAPRGFSCLLLLTGEIDLPVKRRA (HN-S14G), or any sequence setforth in Tables 1-4. In some embodiments humanin or a humaninpolypeptide comprises the sequence: MAPRGFSCLLLLTSEIDLPVKRRA. In someembodiments a humanin analog or humanin polypeptide comprises thesequence: MAPRGFSCLLLLTGEIDLPVKRRA (HN-S14G), or any sequence set forthin Example 1 or Tables 1-4. In some embodiments a humanin polypeptide isselected from one or more of SEQ ID NO:1, SEQ ID NO:2, HNG-F6A, HN-F6A,HN-S7A and HN-C8P. In some embodiments a humanin polypeptide is SEQ IDNO:1. In some embodiments a humanin polypeptide is SEQ ID NO: 2.

In some aspects, the chemotherapeutic agent comprises an alkylatingagent, an anthracycline, an anti-metabolite, plant extract, plantalkaloid, nitrosourea, hormone, nucleoside or nucleotide analog. In someaspects the chemotherapeutic agent comprises a DNA intercalating agentor an agent that attaches or bonds to DNA. In some aspects, thechemotherapeutic agent comprises cyclophosphamide, doxorubicin,azathioprine, cyclosporin A, prednisolone, melphalan, chlorambucil,mechlorethamine, busulphan, methotrexate, 6-mercaptopurine, thioguanine,5-fluorouracil, cytosine arabinoside, 5-azacytidine (5-AZC) and5-azacytidine related compounds, bleomycin, actinomycin D, mithramycin,mitomycin C, carmustine, lomustine, semustine, streptozotocin,hydroxyurea, cisplatin, carboplatin, oxiplatin, mitotane, procarbazine,dacarbazine, a taxane, vinblastine, vincristine, dibromomannitol,gemcitabine, or pemetrexed.

In certain embodiments, a subject has a hyperproliferative disorder. Insome aspects, a subject has a metastatic or non-metastatic neoplasia,tumor, cancer or malignancy. In some embodiments a neoplasia, tumor,cancer or malignancy is metastatic, non-metastatic or benign. In someembodiments a neoplasia, tumor, cancer or malignancy comprises a solidcellular mass. In some embodiments a neoplasia, tumor, cancer ormalignancy comprises hematopoietic cells. In certain embodiments aneoplasia, tumor, cancer or malignancy comprises a carcinoma, sarcoma,lymphoma, leukemia, adenoma, adenocarcinoma, melanoma, glioma,glioblastoma, medulloblastoma, meningioma, neuroblastoma,retinoblastoma, astrocytoma, oligodendrocytoma, mesothelioma,reticuloendothelial, lymphatic or haematopoietic neoplasia, tumor,cancer or malignancy. In certain aspects a sarcoma comprises alymphosarcoma, liposarcoma, osteosarcoma, chondrosarcoma,leiomyosarcoma, rhabdomyosarcoma or fibrosarcoma. In some aspects ahaematopoietic neoplasia, tumor, cancer or malignancy comprises amyeloma, lymphoma or leukemia. In some aspects a neoplasia, tumor,cancer or malignancy comprises a metastatic melanoma. In some aspects aneoplasia, tumor, cancer or malignancy comprises a lung, thyroid, heador neck, nasopharynx, throat, nose or sinuses, brain, spine, breast,adrenal gland, pituitary gland, thyroid, lymph, gastrointestinal (mouth,esophagus, stomach, duodenum, ileum, jejunum (small intestine), colon,rectum), genito-urinary tract (uterus, ovary, cervix, endometrial,bladder, testicle, penis, prostate), kidney, pancreas, liver, bone, bonemarrow, lymph, blood, muscle, or skin, lung, biliary tract, orhematologic neoplasia, tumor, or cancer.

In certain aspects a method herein further comprising administering asecond, third or fourth chemotherapeutic agent.

In some embodiments a humanin or humanin analog is administered priorto, substantially contemporaneously with or following administration ofthe chemotherapeutic agent. In some embodiments a humanin or humaninanalog is administered in combination with the chemotherapeutic agent.

In some aspects a subject has undergone surgical resection,chemotherapy, immunotherapy, ionizing or chemical radiotherapy, local orregional thermal (hyperthermia) therapy, or vaccination. In some aspectsa subject is or is not a candidate for surgical resection, chemotherapy,immunotherapy, ionizing or chemical radiotherapy, local or regionalthermal (hyperthermia) therapy, or vaccination.

Certain embodiments are described further in the following description,examples, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a histogram of the quantity of apoptotic cells in squashedseminiferous tubules (y-axis, mean±SEM) after doxorubicin (DOX), Humanin(HN), Dox and HN (Dox+HN) or no treatment (Con) ex-vivo. Exposing theseminiferous tubules to 43 degrees served as a positive control (Heat).Means with unlike superscripts (e.g., a, b and c as shown above thehistograms) are significantly different (P<0.05).

FIGS. 2A-2B show a histogram of body weight (FIG. 2A, x-axis, grams (g))and testis weight (FIG. 2B, y-axis, grams (g)) of mice 24 hours afterreceiving no treatment (Con, control), HN 40 mg/Kg IP alone (HN),cyclophosphamide (200 mg/Kg IP) alone (CP), or cyclophosphamide andHumanin (CP+HN).

FIGS. 3A-3D show micrograph images of apoptotic cells stained byTerminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), amethod for detecting DNA fragmentation by labeling the terminal end ofnucleic acids. TUNEL positive cells (dark grey or brown in color,arrows) are shown in testis of control treated (FIG. 3A), HN treated(FIG. 3B), cyclophosphamide (CP) treated (FIG. 3C), and combined HN andcyclophosphamide treated mice (FIG. 3D). Scale bar, 100 μm.

FIGS. 4A-D show TUNEL positive apoptotic germ cells (dark grey or brownin color) at stage XII (late stage) of seminiferous epithelial cycle intestis of control (FIG. 4A), HN treated (FIG. 4B), CP treated (FIG. 4C),and combined HN and CP treatment mice (FIG. 4D). Scale bar, 20 μm.

FIG. 4E (Right Panel) shows quantification of germ cell apoptosis atearly/late stages IX-IV as the percentage of tubules with apoptoticcells/100 tubules (y-axis, mean±SEM) in mice after receiving notreatment (Control), Humanin alone (HN), cyclophosphamide alone (CP), orcyclophosphamide and Humanin (CP+HN). Means with unlike uperscripts(e.g., a, b and c as shown above the histograms) are significantlydifferent (P<0.05).

FIGS. 5A-D show TUNEL positive apoptotic germ cells (dark grey or brownin color) at stages VII (middle stage) of seminiferous epithelial cyclein testis of control (FIG. 5A), HN treated (FIG. 5B), CP treated (FIG.5C), and combined HN and CP treatment mice (FIG. 5D). Scale bar, 20 μm.

FIG. 5E (Right Panel) shows quantification of germ cell apoptosis atmiddle stages IX-IV as the percentage of tubules with apoptoticcells/100 tubules (y-axis, mean±SEM) in mice after receiving notreatment (Control), Humanin alone (HN), cyclophosphamide alone (CP), orcyclophosphamide and Humanin (CP+HN). Means with unlike superscripts(e.g., a and b as shown above the histograms) are significantlydifferent (P<0.05).

FIGS. 6A-6B show a histogram of mean body weight (y-axis) of controlmice (FIG. 6A) and mice receiving a tail vein injection of B16 mousemelanoma cells (FIG. 6B). Body weights were measured 3 weeks afterinjection. Two weeks before measuring the body weights, mice weretreated with a humanin analog (HNG), cyclophosphamide (CP) or HNG andcyclophosphamide (CP+HNG) or received no treatment (NT). Means withunlike superscripts (e.g., a and b as shown above the histograms) aresignificantly different (P<0.05).

FIG. 7 shows a histogram of testis weight (y-axis) of mice receiving atail vein injection of B16 mouse melanoma cells. Testis weights weremeasured 3 weeks after injection. Two weeks before measuring the testisweights, mice were treated with HNG (HNG), cyclophosphamide (CP) or HNGand cyclophosphamide (CP+HNG), or received no treatment (NT). Means withunlike superscripts (e.g., a and b as shown above the histograms) aresignificantly different (P<0.05).

FIG. 8 shows a histogram of germ cell apoptosis of mice receiving a tailvein injection of B16 mouse melanoma cells. Germ cell apoptosis wasmeasured 3 weeks after injection. Two weeks before measuring germ cellapoptosis, mice were treated with HNG (HNG), cyclophosphamide (CP) orHNG and cyclophosphamide (CP+HNG), or received no treatment (NT). Germcell apoptosis is expressed as the percent of seminiferous tubulescontaining apoptotic germ cells (y-axis). Means with unlike superscripts(e.g., a, b, c and ab, as shown above the histograms) are significantlydifferent (P<0.05).

FIG. 9 shows micrograph images of metastatic lung melanomas in testis ofmice receiving a tail vein injection of B16 mouse melanoma cells. Testiswere harvested from mice receiving no-treatment (Row NT, images 840NT,841NT, 842NT, 843NT, 844NT) or mice treated with HNG (Row HNG, images824HNG, 821HNG, 823HNG, 820HNG, 822HNG), cyclophosphamide (Row CP,images 830CP, 831CP, 832CP, 833CP, 834CP) or HNG and cyclophosphamide(Row CP+HNG, 825CPHNG, 829CPHNG, 836CPHNG, 839CPHNG, 838CPHNG).

FIG. 10 shows a histogram of the number of lung metastatic tumors(y-axis) in mice receiving a tail vein injection of B16 mouse melanomacells. The number of lung metastatic tumors were counted 3 weeks afterinjection. Two weeks before counting, mice were treated with HNG (HNG),cyclophosphamide (CP) or HNG and cyclophosphamide (CP+HNG), or receivedno treatment (NT). Means with unlike superscripts (e.g., a, b and c, asshown above the histograms) are significantly different (P<0.05).

FIG. 11 shows a histogram of the number of lung metastatic tumors on thesurface of lungs (y-axis) in mice receiving a tail vein injection of B16mouse melanoma cells. The number of lung metastatic tumors were counted3 weeks after injection. Two weeks before counting, mice were treatedwith 5 mg/kg HNG (HNG5), 15 mg/kg HNG (HNG15), cyclophosphamide (CP), 5mg/kg HNG and cyclophosphamide (CPHNG5), 15 mg/kg HNG andcyclophosphamide (CPHNG15), or received no treatment (NT). Means withunlike superscripts (e.g., a, b and c, as shown above the histograms)are significantly different (P<0.05).

FIGS. 12A-12D show micrograph images of the lung metastatic melanomahistology in non-treated (FIG. 12A), HNG (5 mg/kg BW) alone (FIG. 12B),CP alone (FIG. 12C) and CP+HNG (FIG. 12D) treated mice. In the untreatedmice, the undifferentiated melanoma cells were small with mitoticfigures (FIG. 12A); HNG treatment alone did not change the histology ofthe melanoma cells (FIG. 12B); CP treatment induced minordifferentiation of melanoma cells with more cytoplasm (FIG. 12C); andCP+HN treatment resulted marked increase in large differentiated cellswith abundant cytoplasm and presence of melanin in some of these largecells (FIG. 12D).

FIG. 13 shows a histogram of the quantity of apoptotic cells in squashedseminiferous tubules (y-axis, mean±SEM) after ex vivo treatment with lowdose doxorubicin (DOX1), high dose doxorubicin (DOX10), Humanin (HN),low dose Dox1 and HN (Dox1+HN), high dose Dox10 and HN (Dox10+HN) or notreatment (Con) ex-vivo. Exposing the seminiferous tubules to 43 degreesserved as a positive control (Heat). Means with unlike superscripts(e.g., a, b and c as shown above the histograms) are significantlydifferent (P<0.05). The rate of germ cell apoptosis is similar betweencontrol and HN groups (p<0.05). High dose, but not low dose, ofdoxorubicin (DOX) significantly increased the number of apoptotic cellscompared with control (p<0.05). Humanin treatment (HN) significantlyattenuated germ cell apoptosis induced by high dose of DOX treatment(p<0.05).

FIGS. 14A-14B show the effect of HN and HNG on Cyclophosphamide(CP)-induced male germ cell apoptosis. Mice were treated with vehiclecontrol (Control), HN, HNG (Humanin-S14G), CP, CP and HN (CP+HN) or CPand HNG (CP+HNG) as described in experimental procedures (n=4 eachgroup). Apoptotic Index (AI) stands for the ratio of TUNEL positive germcells per Sertoli cell (also in following figures). HN or HNG alone didnot change apoptosis compared with control. CP significantly increasedgerm cell apoptosis both in early+late stages (I-IV and XI-XII, FIG.14A) and middle stages (VII-VIII, FIG. 14B) as compared to control. HNsignificantly suppressed CP-induced apoptosis in early+late stages butnot in middle stages. HNG prevented CP-induce apoptosis both inearly+late and middle stages but fail to reach significance in themiddle stages (P>0.05). Values are means±SEM. Means with unlikesuperscripts (e.g., a, b and c as shown above the histograms) aresignificantly different (P<0.05).

FIGS. 15A-15B show the effect of HN and HNG-F6A on Cyclophosphamide(CP)-induced male germ cell apoptosis. Mice were treated with vehiclecontrol (Control), HN, HNG-F6A (Humanin-S14G-F6A), CP, CP+HN orCP+HNG-F6A as described in experimental procedures (n=4 each group). HNor HNG-F6A alone did not change apoptosis compared with control.Compared with control, CP increased germ cell apoptosis both inearly+late (I-IV and XI-XII, FIG. 15A) and middle (VII-VIII, FIG. 15B)stages. HN significantly suppressed CP-induced apoptosis in early+latestages but not in middle stages. HNG-F6A prevented CP-induce apoptosisboth in early+late and middle stages remarkably. Values are means±SEM.Means with unlike superscripts (e.g., a, b and c as shown above thehistograms) are significantly different (P<0.05).

FIGS. 16A-16D show the effect of HN and HN-S7A on Cyclophosphamide(CP)-induced male germ cell apoptosis and changes of STAT3phosphorylation. Mice were treated with vehicle (Control), HN, HN-S7A,CP, CP+HN, CP+HN-S7A, or CP+HN+HN-S7A as described in experimentalprocedures (n=5 each group). (FIGS. 16A and 16B): Apoptotic cell numberswere determined by TUNEL staining method. HN or HN-S7A alone did notchange apoptosis compared with control. Compared with control, CPincreased germ cell apoptosis both in early+late (I-IV and XI-XII, FIG.16A) and middle (VII-VIII, FIG. 16B) stages. HN significantly suppressedCP-induced apoptosis in early+late stages but not in middle stages.HN-S7A prevented CP-induce apoptosis both in early+late and middlestages remarkably. Addition of HN-S7A to HN did not change the effect ofHN on CP-induced apoptosis. Values are means±SEM. (FIGS. 16C and 16D):Change of STAT3 phosphorylation levels were detected by western blot(FIG. 16C) and presented by density ratio of phosphorylated STAT3/STAT3(FIG. 16D). HN or HN-S7A alone did not change STAT3 phosphorylationcompared with control. Compared with control, CP suppressed STAT3phosphorylation. CP+HN, CP+HN-S7A, or CP+HN+HN-S7A all restored CPsuppressed STAT3 phosphorylation. Density values are means±SEM. Valuesare means±SEM. Means with unlike superscripts (e.g., a and b as shownabove the histograms) are significantly different (P<0.05).

FIGS. 17A-17D show the effect of HN and HN-C8P onCyclophosphamide-induced male germ cell apoptosis and changes of STAT3phosphorylation. Mice were treated with vehicle (Control), HN, HN-C8P,CP, CP+HN, CP+HN-C8P or CP+HN+HN-C8P as described in experimentalprocedures (n=5 each group). (FIGS. 17A and 17B): Apoptotic cell numberswere determined by TUNEL staining method. HN or HN-C8P alone did notchange apoptosis compared with control. Compared with control, CPinduced massive germ cell apoptosis both in early+late (I-IV and XI-XII,FIG. 17A) and middle (VII-VIII, FIG. 17B) stages. CP+HN significantlysuppressed CP-induced apoptosis in early+late stages but not in middlestages. CP+HN-C8P showed weaker preventive effect against CP-induceapoptosis in early+late (p<0.05, compared with CP+HN) but no protectiveeffect in middle stages. Addition of HN-C8P did not change thepreventive effect of HN on CP-induced germ cell apoptosis. Values aremeans±SEM. (FIGS. 17C and 17D): Changes of STAT3 phosphorylation levelswere detected by western blot (FIG. 17C) and presented by density ratioof phosphorylated STAT3/STAT3 (FIG. 17D). HN or HN-C8P alone did notchange STAT3 phosphorylation compared with control. Compared withcontrol, CP suppressed STAT3 phosphorylation. CP+HN, CP+HN-C8P, orCP+HN+HN-C8P all restored CP suppressed STAT3 phosphorylation. Densityvalues are means±SEM. Values are means±SEM. Means with unlikesuperscripts (e.g., a and b as shown above the histograms) aresignificantly different (P<0.05).

FIGS. 18A-18B show the effect of HN and HN-L12A onCyclophosphamide-induced male germ cell apoptosis. Mice were treatedwith vehicle (Control), HN, HN-L12A, CP, CP+HN, CP+HN-L12A, orCP+HN+HN-L12A as described in experimental procedures (n=5 each group).Apoptotic cell numbers were determined by TUNEL staining method. HN orHN-L12A alone did not change apoptosis compared with control. Comparedwith control, CP increased germ cell apoptosis both in early+late (I-IVand XI-XII, FIG. 18A) and middle (VII-VIII, FIG. 18B) stages. CP+HNsignificantly suppressed CP-induced apoptosis in early+late stages butnot in middle stages. CP+HN-L12A showed no preventive effect againstCP-induced apoptosis in neither early+late or middle stages. CP+HN'spreventive effect was completely blocked by CP+HN+HN-L12A. Values aremeans±SEM. Means with unlike superscripts (e.g., a, b and c as shownabove the histograms) are significantly different (P<0.05).

FIG. 19 shows HNG enhanced CP-induced suppression of mammary tumor. Micebearing 4T1 mammary cancer were treated for 14 days with HNG (5mg/kg/day), CP (100 mg/kg), HNG+CP or nothing (NT). Mammary tumor weightis shown on the y-axis.

FIG. 20 shows a photomicrograph of ovaries obtained from young mice(control), 4T1 mammary cancer bearing mice were not treated, or treatedwith HNG (5 mg/kg/day), CP (100 mg/kg IP), HNG+PC (Hematoxyline & Eosin,scale bar=100 micrometers).

FIG. 21 shows blood levels (y-axis) of glucose, IGF-1, IGFBP-3 andIGFBP-1 in 30-week-old mice before (Control) or after 72 hours fasting(72h-STS).

FIG. 22 shows plasma levels of IGF-1 (y axis) in mice bearing metastaticlung melanomas (NT not treated; or treated for 14 days with HNG 5mg/kg/day IP, CP 200 mg/kg IP single injection; or HNG+CP).

FIG. 23 shows a proposed theory of HN action in cancer where HN mimicscaloric restriction and decreases IGF-1 inhibiting downstream signalingof PI3K-Akt-mTOR and RAS-RAF-MEK leading to suppression ofproliferation, survival, and metastasis.

FIG. 24 shows apoptosis of germ cells in mice (apoptotic index, y-axis)treated with HNG, CP, or CP and HNG (CP+HNG). HNG decreased CP-inducedgerm cell apoptosis in mice. Apoptotic index: # apoptotic cells/Sertolicell.

FIG. 25 shows the sperm count (y axis) in Cauda Epididymis from micetreated with repeated CP, HNG or CP and HNG injections (CP+HNG) comparedto untreated controls (Con).

FIG. 26 shows tumor weight of human medulloblastoma (DAOY) tumorsremoved 26 days after implantation into the right flank of SCID micetreated with nothing (control), HNG, Temozolomide (TMZ) or TMA and HNG(TMZ+HNG).

FIGS. 27A-27B show the effect of HN and HN-L12A on TMZe-induced malegerm cell apoptosis in middle stages (FIG. 27A) and early-late stages(FIG. 27B). Mice were treated with vehicle (Control), HNG, TMZ or TMZand HNG (TMZ+HNG) as described in experimental procedures (n=5 eachgroup). Apoptotic cell numbers were determined by TUNEL staining method.HNG alone had no effect on male germ cell apoptosis, but significantlyprevented TMZ-induced apoptosis in both middle stages (FIG. 27A) andearly+late stages (FIG. 27B). Values are means±SEM. Means with unlikesuperscripts (e.g., a and b as shown above the histograms) aresignificantly different (P<0.05).

FIGS. 28A-28B show a histogram of intratesticular (FIG. 28A) and serumtesticular (FIG. 28B) testosterone (T) levels in animals treated withEDS (Black Bars) or not pre-treated with EDS (Light grey bars). In theEDS treated animals, intratesticular and serum testosterone levels weremarkedly reduced in all groups (EDS, EDS+HN, EDS+CP, EDS+CP+HN). HN didnot restore intratesticular and serum testosterone levels in the EDStreated rats (Black bars). There were 4 rats in each group.

FIGS. 29A-29B show shows Apoptotic Index [% of cross sections ofseminiferous tubules (ST) containing TUNEL positive germ cells/totalcross sections of seminiferous tubules](y axis) at early (I-VI) and late(IX-XIV) stages of seminiferous epithelium cycle (FIG. 29A). In thegroups of rats with the presence of Leydig cells (light bars), CPincreased apoptosis compared to vehicle (p=0.0019, dashed bracket)control, and HN reduced the CP-induced apoptosis (p=0.021). EDSpre-treatment (dark bars) had a minimal effect on apoptosis of germcells (p=0.169), and HN had no significant effect on apoptosis in theEDS (p=0.251) (FIG. 29A, dark bars) at early and late stages. FIG. 29Bshows Apoptotic Index (y axis) at middle (VII-VIII) stages ofseminiferous epithelium cycle. CP did not increase apoptosis in themiddle stages in animals not pre-treated with EDS (FIG. 29B. lightbars). In the EDS pre-treated rats (dark bars), EDS increased apoptosisof germ cells compared to control (p=0.0014, dashed bracket) and HNdecreased apoptosis levels in EDS treated rats (p=0.018). Addition of CPto EDS did not further increased germ cell apoptosis. CP+EDS increasedapoptosis compared to control (p=0.0011, dashed bracket) and HNprevented apoptosis EDS+CP treated rats (p=0.04) (FIG. 29B. Dark bars).There were 4 rats in each group.

FIG. 30 shows the effect of HN on testosterone levels of Leydig cellsexposed to KTZ for 4 hours in vitro. KTZ markedly reduced testosteronelevels in cultured Leydig cells. HN did not restore testosterone levelssuppressed by KTZ to baseline. Eight replicate experiments wereperformed where culture Leydig cells were treated with vehicle(control), HN, KTZ, and HN+KTZ respectively.

The drawings illustrate embodiments of the technology and are notlimiting. For clarity and ease of illustration, the drawings are notmade to scale and, in some instances, various aspects may be shownexaggerated or enlarged to facilitate an understanding of particularembodiments.

DETAILED DESCRIPTION

Described herein are compositions comprising humanin and/or one or morehumanin analogs and method of use thereof. In some embodiments acomposition comprises a humanin polypeptide and/or a polypeptide of ahuman analog. In some embodiments a composition described hereincomprises one or more polypeptides comprising a humanin polypeptideand/or a polypeptide of a human analog. Humanin and/or a humanin analogor composition thereof can be incorporated into pharmaceuticalcompositions, e.g., a composition comprising a pharmaceuticallyacceptable carrier and/or excipient. Such pharmaceutical compositionsare useful for, among other things, administration and/or delivery to asubject.

Humanin (HN) is a 24-amino acid mitochondrial derived peptide with theamino acid sequence of SEQ ID NO:1 (HN, humanin polypeptide). Humanin isan endogenous peptide found in many tissues including neurons(Hashimoto, et al., 2001; Hashimoto, et al., 2001), endothelial cells(Bachar, et al., 2010), pancreatic beta cells (Hoang, et al., 2010), andcardiomyocytes (Muzumdar, et al., 2010). HN is expressed in germ cellsand Leydig cells in testes (Colon, et al., 2006; Moretti, et al., 2010).HN reportedly protects against male germ cell apoptosis induced bytesticular hormonal deprivation (Jia, et al., 2013; Lue, et al., 2010).In addition to the finding of endogenous HN (peptide or gene) in normaltissues and cells, HN has been proposed as an potential oncopeptide(Maximov, et al., 2002) because HN gene is expressed in cutaneous T-celllymphoma (Hartmann, et al., 2008), diffuse large B-cell lymphoma(Tarantul & Hunsmann, 2001), and gastric cancer (Mottaghi-Dastjerdi, etal., 2014).

In some embodiments, humanin comprises the amino acid sequence of SEQ IDNO: 1. A humanin analog can be a humanin variant or derivative thereof.Non-limiting examples of humanin, humanin analogs and/or variantsapplicable to the methods, uses and compositions set forth herein areshown and described in Example 1 and Tables 1-4. Humanin and humaninanalogs (i.e., collectively referred to as humanin polypeptides), and/orderivatives thereof are contemplated for compositions and methodsdescribed herein. A derivative of humanin or a derivative of a humaninanalog may comprise any suitable amino acid modification, and/or maycomprise amino acid analogs or conjugates. For example, a derivative ofhumanin or humanin analogs may be conjugated to a label, an antibody, atag, a carrier protein, a ligand, the like or combinations thereof.

As described herein, compositions comprising humanin and/or one or morehumanin analogs can be used to alter the effects of chemotherapeuticagents. As disclosed herein, HN and HN analogs can be used as agents toprotect germ cells from death and/or damage caused by chemotherapeuticagents that are sometimes used for treatment of cancer and tumors. Forexample, as shown herein HN and HN analogs are able to protect animalsagainst chemotherapy induced germ cell apoptosis by cyclophosphamide(CP), doxorubicin (dox) and/or temozolomide (TMZ) treatment. In someembodiments, HN and/or HN analogs can be used to enhance chemotherapyinduced tumor cell cytotoxicity. For example, as shown herein, HN and/orHN analogs can enhance tumor cell apoptosis induced by cyclophosphamide(CP), doxorubicin (dox) and/or Temozolomide (TMZ) treatment. Thesediscoveries are clinically relevant, as methods, uses and compositionsdescribed herein can be used as an adjunct to treatments of certainhyperproliferative disorders, (e.g., neoplasias, tumors, cancers andmalignancies) in which germ cell development is suppressed and/or wheregerm cells are killed by treatment with chemotherapy. In certainembodiments, method and compositions herein can protect a cancer patientfrom many common adverse effects of chemotherapy and/or enhance thedesired anti-tumor effects of a chemotherapeutic agent.

Methods, uses and compositions herein are applicable to any subject. Theterm “subject” refers to animals, typically mammalian animals. Anysuitable mammal can be treated by a method described herein.Non-limiting examples of mammals include humans, non-human primates(e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, andthe like), domestic animals (e.g., dogs and cats), farm animals (e.g.,horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse,rat, rabbit, guinea pig). A mammal can be any age or at any stage ofdevelopment (e.g., an adult, teen, child, infant, or a mammal in utero).A mammal can be male or female. A mammal can be a pregnant female. Incertain embodiments a mammal can be an animal disease model, forexample, animal models used for the study of viral infections. In someembodiments a mammal is a human. In some embodiments a mammal is a humanmale. In some embodiments a mammal is a human female.

A patient can be any subject suspected of having, diagnosed with,suspected of having, or undergoing treatment for an ailment, disease orinfection, or a subject who could benefit from a use or method herein.In certain embodiments a subject has a hyperproliferative disorder(e.g., a cancer) and/or is undergoing a treatment for ahyperproliferative disorder. A subject can have any type ofhyperproliferative disorder. In some embodiments, a subject has, or issuspected of having a hyperproliferative disorder thought to betreatable by a chemotherapeutic agent.

In some embodiments a subject is undergoing, has undergone, or willundergo a treatment for a hyperproliferative disorder. In someembodiments a subject is a candidate for a treatment for ahyperproliferative disorder. Non-limiting examples of a treatment for ahyperproliferative disorder include a surgical resection, chemotherapy,targeted therapy (e.g., immunotherapy, antibody therapy), ionizingradiotherapy (e.g., radiation therapy, proton therapy and the like),chemical radiotherapy, local or regional thermal (hyperthermia) therapy,hormonal therapy, adjuvant therapy, a vaccination, the like, orcombinations thereof.

In certain embodiments a subject is undergoing, has undergone or willundergo chemotherapy. In certain embodiments a subject is a candidatefor a chemotherapy treatment. Chemotherapy comprises administration ofone or more chemotherapeutic agents to a subject. A chemotherapeuticagent may be self-administered or administered by another (e.g., by ahealth care professional). A chemotherapeutic agent can be administeredby any suitable method (e.g., by any suitable route, at any suitabledose, for any suitable amount of time (e.g., continuously, periotically,and the like)).

In some embodiments a subject is not undergoing or has not undergone atreatment for a hyperproliferative disorder. In some embodiments asubject is not a candidate for one or more treatments for ahyperproliferative disorder, non-limiting examples of which include asurgical resection, targeted therapy (e.g., immunotherapy, antibodytherapy), ionizing radiotherapy (e.g., radiation therapy, proton therapyand the like), chemical radiotherapy, local or regional thermal(hyperthermia) therapy, hormonal therapy, adjuvant therapy, avaccination, the like, or combinations thereof. In some embodiments asubject is not a candidate for chemotherapy.

In some embodiments a hyperproliferative disorder is characterized ornamed according to the presence of a hyperproliferative tissue or celltype. Non-limiting examples of a hyperproliferative disorder and/orhyperproliferative tissue/cell types include melanoma, lymphoma (e.g.,Hodgkin lymphoma, non-Hodgkin lymphoma, a B-cell neoplasm, a T-cellneoplasm, an NK cell neoplasm), leukemia, reticuloendothelialhyperplasia (e.g., reticuloendothelial neoplasia), lymphatic neoplasia,hematopoietic neoplasia, myeloma, multiple myeloma, animmunodeficiency-associated lymphoproliferative disorder, adenoma,adenocarcinoma, sarcoma (non-limiting examples of which include alymphosarcoma, liposarcoma, osteosarcoma, chondrosarcoma,leiomyosarcoma, rhabdomyosarcoma, fibrosarcoma, the like or combinationsthereof), carcinoma, breast cancer, colorectal cancer, gastrointestinalcancer, hepatocellular cancer, lung cancer, bone cancer, renal cancer,bladder cancer, hepatoma, neuroblastoma, retinoblastoma, astrocytoma,glioma, glioblastoma, medulloblastoma, meningioma, oligodendrocytoma,cervical cancer, testicular cancer, ovarian cancer, mesothelioma,esophageal cancer, pancreatic cancer, prostate cancer, the like orcombinations thereof. A hyperproliferative disorder may be benign,malignant, metastatic, non-metastatic or undetermined.

In some embodiments a neoplasia, tumor, cancer or malignancy comprises ametastatic melanoma. In some embodiments a neoplasia, tumor, cancer ormalignancy comprises a lung, thyroid, head or neck, nasopharynx, throat,nose or sinuses, brain, spine, breast, adrenal gland, pituitary gland,thyroid, lymph, gastrointestinal (mouth, esophagus, stomach, duodenum,ileum, jejunum (small intestine), colon, rectum), genito-urinary tract(uterus, ovary, cervix, endometrial, bladder, testicle, penis,prostate), kidney, pancreas, liver, bone, bone marrow, lymph, blood,muscle, or skin, lung, biliary tract, or hematologic neoplasia, tumor,or cancer. In some embodiments a neoplasia, tumor, cancer or malignancycomprises a solid cellular mass. In certain embodiments a malignantneoplasm comprises or consist of a fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteosarcoma, chordoma, malignant fibroushistiocytoma, hemangiosarcoma, angiosarcoma, lymphangiosarcoma,mesothelioma, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma,epidermoid carcinoma, malignant skin adnexal tumor, adenocarcinoma,hepatoma, hepatocellular carcinoma, renal cell carcinoma, hypernephroma,cholangiocarcinoma, transitional cell carcinoma, choriocarcinoma,seminoma, embryonal cell carcinoma, glioma, glioblastoma multiforme,neuroblastoma, medulloblastoma, malignant meningioma, malignantschwannoma, neurofibrosarcoma, parathyroid carcinoma, medullarycarcinoma of thyroid, bronchial carcinoid, oat cell carcinoma, malignantpheochromocytoma, islet cell carcinoma, malignant carcinoid,retinoblastoma, chemodectoma, paraganglioma, malignant carcinoid,malignant paraganglioma, melanoma, malignant schwannoma, merkel cellneoplasm, cystosarcoma phylloides, wilms tumor, malignant ovariantumors, malignant testicular tumors, the like, or combinations thereof.In certain embodiments a neoplasia, tumor, cancer or malignancycomprises a carcinoma, sarcoma, lymphoma, leukemia, adenoma,adenocarcinoma, melanoma, glioma, glioblastoma, medulloblastoma, Kaposisarcoma, meningioma, neuroblastoma, retinoblastoma, astrocytoma,oligodendrocytoma, reticuloendothelial, lymphatic or haematopoieticneoplasia, tumor, cancer or malignancy. In certain embodiments a sarcomacomprises a lymphosarcoma, liposarcoma, osteosarcoma, chondrosarcoma,leiomyosarcoma, rhabdomyosarcoma or fibrosarcoma.

In certain embodiments a neoplasia, tumor, cancer or malignancycomprises a metastatic melanoma. In certain embodiments a neoplasia,tumor, cancer or malignancy comprises a lung, thyroid, head or neck,nasopharynx, throat, nose or sinuses, brain, spine, breast, adrenalgland, pituitary gland, thyroid, lymph, gastrointestinal (mouth,esophagus, stomach, duodenum, ileum, jejunum (small intestine), colon,rectum), genito-urinary tract (uterus, ovary, cervix, endometrial,bladder, testicle, penis, prostate), kidney, pancreas, liver, bone, bonemarrow, lymph, blood, muscle, or skin, lung, biliary tract, orhematologic neoplasia, tumor, or cancer.

In some embodiments a leukemia is an acute lymphocytic leukemia (ALL),acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL),chronic myeloid leukemia (CML), or chronic myelomonocytic leukemia(CMML).

Any suitable chemotherapeutic agent can be administered to a subject.Chemotherapeutic agents are often administered to a subject (e.g., apatient) for the treatment of a hyperproliferative disease or disorder.Chemotherapeutic agents can include a variety of poisons, venoms,toxins, proteins, antibodies and inhibitors that can induce suppressionof cellular activities and/or death of a mammalian cell by a variety ofmechanisms, non-limiting examples of which include apoptosis (programmedcell death), activation-induced cell death, autophagy, necrosis,necroptosis, and the like). Cell death and/or viability can bedetermined by a suitable assay known in the art or described herein,non-limiting examples of which include a suitable membrane alterationassay (e.g., as measured by annexin-V binding, uptake of impermeabledyes such as propidium iodide, trypan blue, LDH release, the like orcombinations thereof), caspase activation assays (e.g., as measured bypeptide substrate cleavage, substrate cleavage (e.g., PARP, M30),caspase processing, the like or combinations thereof), DNA fragmentationassays (e.g., TUNEL assay, or assessment of DNA laddering, cytoplasmicnucleosomes, hypodipoloid DNA, and release of incorporated nucleotides(e.g., BrdU), the like, or combinations thereof), mitochondrial damageassays (e.g., measurements of cytochrome C release, mitochondrialmembrane potential, ATP production, electron transport activity (e.g.,WST-1 or MTT assays)), the like or combinations thereof.

In certain embodiments a cellular activity is suppressed by achemotherapeutic agent. Non-limiting examples of cellular activitiesthat may be suppressed by a therapeutic agent include growth, mitosis,meiosis, motility, respiration, maturation, differentiation,transcription, translation, DNA replication, mitochondrial respiration,certain catabolic or metabolic activities, secretion, endocytosis,phagocytosis, the like, or combinations thereof. In some embodiments,suppression and grammatical variations thereof, refers to an adverseeffect of a chemotherapeutic agenton a cell that results in theinhibition, reduction or loss of one or more functions of the cell. Insome embodiments the inhibition, reduction or loss of one or more cellfunctions refers to the loss of, or inhibition of, a cell's ability toreplicate (e.g., proliferate) and/or undergo mitosis or meiosis. In someembodiments the inhibition, reduction or loss of one or more cellfunctions refers to the loss of, or inhibition of, a cell's ability tometabolize oxygen, proteins, fatty acids, carbohydrates and/or glucose.In some embodiments the inhibition, reduction or loss of one or morecell functions refers to the loss of, or inhibition of, a cell's abilityto initiate, carry out, maintain or terminate an adaptive or innateimmune response, or a portion thereof. In some embodiments theinhibition, reduction or loss of one or more cell functions refers tothe loss of, or inhibition of, a cell's ability to initiate, carry out,maintain or terminate an immune function, non-limiting examples of whichinclude antigen presentation; apoptosis; phagocytosis; pinocytosis;T-cell activation; B-cell activation; expressing, presenting, secretingand/or responding to an antigen, cytokine, chemokine, growth factors,TNF or TNF-related family members, interferon, porin, defensin,complement, protease, antibody, a hormone, and/or receptors thereof; thelike; or combinations thereof.

Suppression and/or death of one or more cells can be induced by achemotherapeutic agent. Suppression and/or death of cells can be inducedwhen a cell comes into contact with one or more chemotherapeutic agents.In some embodiments a chemotherapeutic agent is cytotoxic to a cell. Incertain embodiments, administration of a chemotherapeutic agent to asubject induces, promotes, increases and/or stimulates suppressionand/or death of germ cells in the absence of a method described herein(e.g., in the absence of administering humanin or a humanin analog). Incertain embodiments, administration of a therapeutic agent to a subjectreduces, decreases, or inhibits maturation, proliferation and/orsurvival of germ cells in the absence of a method described herein(e.g., in the absence of administering humanin or a humanin analog). Incertain embodiments, administration of a chemotherapeutic agent to asubject damages germ cells in the absence of a method described herein(e.g., in the absence of administering humanin or a humanin analog).Cell damage may include damage to genomic DNA, mitochondria or otherorganelles, mitochondrial DNA, mitochondrial cell walls or phospholipidmembranes.

In some embodiments a chemotherapeutic agent comprises a cytotoxiccompound. In some embodiments a chemotherapeutic agent comprises orconsists of one or more cytotoxic compounds. Cytotoxic compounds can beorganic or inorganic compounds. In some embodiments cytotoxic compoundsare relatively small compounds with a molecular weight between 1 andabout 20,000 Daltons, 1 and about 10,000 Daltons, 1 and about 5000Daltons, 1 and about 2500 Daltons, 1 and about 1000 Daltons, 1 and about500 Daltons or between about 50 and about 1000 Daltons.

In some embodiments a chemotherapeutic agents is a protein orpolypeptide. In some embodiments a chemotherapeutic agents is anantibody (e.g., a monoclonal or polyclonal antibody). Chemotherapeuticagents can be polypeptides or fusion proteins. In some embodiments,chemotherapeutic agents are not cytotoxic until after they areadministered to a subject wherein the chemotherapeutic agents aremetabolized into a cytotoxic compound (e.g., cyclophosphamide). In someembodiments a cell is contacted with a chemotherapeutic agent and thecell metabolizes the chemotherapeutic agent into a cytotoxic compound. Acell can be contacted directly or indirectly (e.g., by a targetedapproach) with a chemotherapeutic agent. In some embodiments achemotherapeutic agents is autoimmune chemotherapeutic agents fortreatment of an autoimmune disorder.

In some embodiments a chemotherapeutic agent comprises or consists of analkylating agent, an anthracycline, cytoskeletal disruptors, epothilones(e.g., epothilone), histone deacetylase inhibitors (e.g., vorinostat,romidepsin), inhibitors of topoisomerase I (e.g., irinotecan,topotecan), inhibitors of topoisomerase II (e.g., etoposide, teniposide,tafluposidean), kinase inhibitors, peptide antibiotics (e.g., bleomycin,actinomycin), platinum-based agents (e.g., carboplatin, cisplatin,oxaliplatin), retinoids (e.g., tretinoin, alitretinoin, bexarotene),vinca alkaloids and derivatives (e.g., vinblastine, vincristine,vindesine, vinorelbine), anti-metabolites, plant extracts, plantalkaloids, nitrosourea, hormone, nucleoside or nucleotide analog andcombinations thereof.

In some embodiments a chemotherapeutic agent comprises an alkylatinganti-neoplastic agent (e.g., an alkylating anti-neoplastic agent. Analkylating antineoplastic agent is a class of chemotherapeutic agentsthat work, in part, by attaching an alkyl group (e.g., C_(n)H_(2n+1)) toDNA, a process known alkylation. Some alkylating antineoplastic agentsare administered as a pro-drug that is converted in vivo to an activealkylating agent. An alkylating antineoplastic agent often alkylates aguanine base of DNA. Alkylating antineoplastic agents are most effectiveon proliferating cells (e.g., cancer cells) which, in general,proliferate faster and with less error-correcting than healthy cells.Without being limited to theory, it is thought that proliferating cellsare more sensitive to DNA damage (e.g., alkylation), which ofteninitiates a cell death pathway (e.g., apoptosis). However, Alkylatingantineoplastic agents can also be toxic to normal cells (cytotoxic), inparticular in cells that divide frequently, such as those in thegastrointestinal tract, bone marrow, and germ cells of the testicles andovaries. Non-limiting examples of alkylating anti-neoplastic agentsAltretamine (hexamethylmelamine, HEXALEN®), Busulfan, Carmustine (BCNU),Chlorambucil, Cyclophosphamide, Dacarbazine (DTIC), Ifosfamide,Lomustine (CCNU), Mechlorethamine, Melphalan, Procarbazine,Streptozotocin, Temozolomide, Thiotepa (triethylenethio-phosphoramide),Carboplatin, Cisplatin, Oxaliplatin, the like or combinations thereof.In some embodiments a chemotherapeutic agent comprises Altretamine(hexamethylmelamine, HEXALEN®), Busulfan, Carmustine (BCNU),Chlorambucil, Cyclophosphamide, Dacarbazine (DTIC), Fotemustine,Ifosfamide, Lomustine (CCNU), Mechlorethamine, Melphalan, Procarbazine,semustine (MeCCNU), Streptozotocin, Temozolomide, Thiotepa(triethylenethio-phosphoramide), Carboplatin, Cisplatin, and/orOxaliplatin, monofunctional alkylators, nitrosoureas, temozolomide, thelike, analogs or combinations thereof.

In some embodiments a chemotherapeutic agent comprises a DNAintercalating agent which is often an agent that attaches or bonds toDNA or RNA. In some embodiments a DNA intercalting agent comprises ananthracycline. In some embodiments a DNA intercalating agent comprisesor consists of acrolein, phosphoramide, Actinomycin D, bleomycin,idarubicin, daunorubicin, doxorubicin, elsamicin A, epirubicin,ethidium, m-AMSA, mitoxantrone, doxorubicin (Adriamycin, Doxil, Myocet,hydroxydaunorubicin, hydroxydaunomycin), Epirubicin, Idarubicin,Valrubicin, TAS-103, MLN944 (XR5944), Obatoclax, mechlorethamine,methotrexate, 6-mercaptopurine, thioguanine, 5-fluorouracil, cytosinearabinoside, 5-azacytidine (5-AZC) and 5-azacytidine related compounds,mithramycin, mitomycin C, hydroxyurea, carboplatin, oxiplatin, mitotane,a taxane, vinblastine, vincristine, dibromomannitol, gemcitabine,pemetrexed, the like or a combination thereof. In some embodiments a DNAintercalating agent comprises or consists of Actinomycin D, bleomycin,daunorubicin, doxorubicin, elsamicin A, epirubicin, ethidium, m-AMSA,mitoxantrone, doxorubicin (Adriamycin, Doxil, Myocet,hydroxydaunorubicin, hydroxydaunomycin), Epirubicin, Idarubicin,Valrubicin, TAS-103, MLN944 (XR5944), and Obatoclax.

In some embodiments a chemotherapeutic agent comprises a cytoskeletaldisruptor. Non-limiting examples of cytoskeletal disruptors (e.g.,taxanes) include paclitaxel, taxol, and docetaxel.

In some embodiments a chemotherapeutic agent comprises a kinaseinhibitor. Non-limiting examples of kinase inhibitors includebortezomib, erlotinib, gefitinib, imatinib, vemurafenib, vismodegib, thelike, analogs and derivatives thereof.

In some embodiments a chemotherapeutic agent comprises one or morenucleotide analogs. Non-limiting examples of nucleotide analogs includeazacitidine, azathioprine, capecitabine, cytarabine, doxifluridine,fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate,tioguanine (formerly thioguanine), the like, analogs and derivativesthereof.

In some embodiments a chemotherapeutic agent comprises one or moredifferent chemotherapeutic agents. In some embodiments achemotherapeutic agent comprises a cocktail of chemotherapeutic agentscomprising two, three, four, five or more chemotherapeutic agents.

In some embodiments a chemotherapeutic agent induces partial or completedestruction of some or all hyperproliferative cells in a subject. Insome embodiments a chemotherapeutic agent induces partial or completedestruction of a neoplastic, tumor, cancer or malignant cell mass in asubject. A chemotherapeutic agent can decrease the volume or size of aneoplasia, neoplastic tumor, cancer or malignancy and/or reduce thenumbers of hyperproliferative cells in a subject. In some embodiments achemotherapeutic agent stimulates and/or induces apoptosis, necrosis,and/or lysis of hyperproliferative cells or cells of a neoplastic tumor,cancer or malignant cell masses in a subject. In some embodiments achemotherapeutic agent inhibits or prevents progression of or anincrease in hyperproliferative cells or a neoplasia, tumor, cancer ormalignancy. In some embodiments a chemotherapeutic agent prolongslifespan of a subject comprising a hyperproliferative disease ordisorder. The efficacy or activity of a chemotherapeutic agent can bedetermined according to one or more of 1) its ability and effectivenessto induce partial or complete destruction of some or allhyperproliferative cells in a subject, 2) induce partial or completedestruction of a neoplastic, tumor, cancer or malignant cell mass in asubject, 3) decrease the volume or size of a neoplasia, neoplastictumor, cancer or malignancy and/or reduce the numbers ofhyperproliferative cells in a subject, 4) stimulate and/or inducesapoptosis, necrosis, and/or lysis of hyperproliferative cells or cellsof a neoplastic tumor, cancer or malignant cell masses in a subject, 5)inhibit or prevent progression of or an increase in hyperproliferativecells or a neoplasia, tumor, cancer or malignancy in a subject, and/or6) prolong the lifespan of a subject comprising a hyperproliferativedisease or disorder. In certain embodiments, the administration ofhumanin or a humanin analog does not substantially reduce, decrease,suppress or inhibit efficacy or activity of a chemotherapeutic agent. Insome embodiments, the administration of humanin or a humanin analogenhances, increases or substantially increases efficacy or activity of achemotherapeutic agent.

A subject often comprises one or more germ cells. A germ cell can be anycell that is, or is destined to become, a sperm or egg. A germ cell canbe diploid or haploid. In some embodiments a germ cell is haploid. Insome embodiments a germ cell refers to a gametogonia (diploid), primarygametocyte (diploid), secondary gametocyte (haploid), gametid (haploid),and/or a gamete (haploid). In some embodiments a germ cell refers to aspermatogonia (diploid), spermatocyte, spermatid, spermatozoa, and/or asperm cell. In some embodiments a germ cell refers to Oogonia (diploid),Oocyte (e.g., or a follicle containing an oocyte), and/or Ovum (eggcell). In some embodiments a germ cell is destined to undergo a processof oncogenesis or spermatogenesis. In some embodiments a germ cell is asperm. In some embodiments a germ cell is an oocyte or ovum.

Administration of a chemotherapeutic agent to a subject can reduce theamount of viable germ cells (e.g., mature sperm, mature ovum or oocytes)in a subject thereby reducing or decreasing fertility in a subject. Thisis one of the deleterious effects (e.g., adverse effects) ofadministering a chemotherapeutic agent. Without being limited to theory,a chemotherapeutic agent may reduce the amount of germ cells byinhibiting or suppressing oogenesis and/or spermatogenesis, byinhibiting mitosis or meiosis of germ cells, and/or by inducing celldeath of germ cells.

In some embodiments a method, use or composition described hereinprotects germ cells in a subject from suppression and/or death inducedby a chemotherapeutic agent. In some embodiments a method, use orcomposition described herein inhibits and/or reduces germ cellsuppression and/or death induced by a chemotherapeutic agent. Withoutbeing limited by theory, a method, use or composition described hereinmay protect germ cells by preserving germ cell viability and/or functionof one or more germ cells from the deleterious effects (e.g., adverseeffects) caused by administration of a chemotherapeutic agent. In someembodiments a method, use or composition described herein protects germcells by inhibiting or blocking chemotherapy-induced (i.e., induced byadministration of a chemotherapeutic agent) cell death of germ cells. Amethod, use or composition described herein may reduce or inhibit thecytotoxic effects of a chemotherapeutic agent on a germ cell, regardlessof the mechanism of cytotoxicity. In some embodiments a method, use orcomposition described herein protects germ cells by inhibitingchemotherapy-induced apoptosis, necrosis, activation-induced cell death,autophagy, and/or necroptosis of germ cells. In some embodiments amethod, use or composition described herein protects germ cells byinhibiting chemotherapy-induced apoptosis of germ cells. In certainembodiments a method, use or composition described herein may inhibitcertain signaling pathways that may lead to apoptosis where theapoptotic pathway is activated by a chemotherapeutic agent. In someembodiments a method, use or composition described herein protects germcells from chemotherapy-induced DNA damage (e.g., alkylation,cross-linking, intercalation and the like).

In some embodiments, a method of protecting germ cells reduces germ celldeath induced by a chemotherapeutic agent by at least 1000%, 500%, atleast 200%, at least 150%, at least 100%, at least 50%, at least 30%, atleast 20%, at least 15%, at least 10%, or at least 5%, therebyprotecting germ cells from cell death. In some embodiments, a method ofprotecting germ cells described herein increases the number of viablegerm cell by at least 1000 fold, 500 fold, at least 200 fold, at least150 fold, at least 100 fold, at least 50 fold, at least 30 fold, atleast 20 fold, at least 15 fold, at least 10 fold, at least 5 fold, orby at least 2 fold, thereby protecting germ cells fromchemotherapy-induced cell death. In some embodiments, a method ofprotecting germ cells described herein maintains the number of viablegerm cells during or after a chemotherapeutic treatment within +/−50%,+/−40%, +/−30%, +/−20%, or +/−10% of the number of viable germ cellsexisting prior to a chemotherapeutic treatment, thereby protecting germcells from a chemotherapeutic treatment (e.g., chemotherapy-induced celldeath).

Administration of a chemotherapeutic agent to a subject can result in adecrease or reduction of fertility of a male or female subject.Administration of a chemotherapeutic agent to a subject can result ininfertility of a male or female subject. In some embodiments, acomposition or method herein inhibits a decrease or reduction offertility in a subject caused by treatment with a chemotherapeuticagent.

In some embodiments, a composition or method herein inhibits a decreaseor reduction of fertility in a subject caused by treatment with achemotherapeutic agent. In some embodiments, a composition or methodherein inhibits a chemotherapy-induced decrease or reduction offertility by at least 1000%, 500%, at least 200%, at least 150%, atleast 100%, at least 50%, at least 30%, at least 20%, at least 15%, atleast 10%, or by at least 5%. In some embodiments, a composition ormethod herein substantially inhibits a chemotherapy-induced decrease orreduction of fertility where fertility in a chemotherapy treated subjectis maintained within +/−50%, +/−40%, +/−30%, +/−20%, or +/−10% of thefertility of the subject as measured or estimated prior to achemotherapy treatment. Methods of measuring or estimating fertility ofa male or female subject are known in the art.

In certain embodiments a method, use or composition described hereinreduces or inhibits germ cell suppression (e.g., suppression ofproliferation) induced by a chemotherapeutic agent by at least 200%, atleast 150%, at least 100%, at least 50%, at least 30%, at least 20%, atleast 15%, at least 10%, or by at least 5%.

In some embodiments a method, use or composition described hereindecreases germ cell suppression and/or death. In some embodiments amethod, use or composition described herein decreases germ cellsuppression or death induced by a chemotherapeutic agent. In someembodiments a method, use or composition described herein decreases germcell death induced by a chemotherapeutic agent by up to 100%, up to 50%,up to 30%, up to 20%, up to 15%, up to 10%, or up to 5%. In certainembodiments a method, use or composition described herein decreases germcell suppression (e.g., suppression of proliferation) induced by achemotherapeutic agent by at least 200%, at least 150%, at least 100%,at least 50%, at least 30%, at least 20%, at least 15%, at least 10%, orby at least 5%.

In some embodiments a method, use or composition described hereinpromotes and/or increases maturation, proliferation and/or survival ofgerm cells in a subject. In some embodiments a method, use orcomposition described herein promotes and/or increases maturation,proliferation and/or survival of germ cells in a subject that wasadministered a chemotherapeutic agent. In some embodimentsadministration or delivery of humanin or a humanin analog promotesand/or increases maturation, proliferation and/or survival of germ cellsin a subject that was administered a chemotherapeutic agent. In certainembodiments, administration of a chemotherapeutic agent (e.g., acytotoxic compound) to a subject reduces, decreases, or inhibitsmaturation, proliferation and/or survival of germ cells in the absenceof administration or delivery of humanin or humanin analog, whichreduction, decrease or inhibition can be reversed completely orpartially by administration of humanin or a humanin analog. In someembodiments a method, use or composition described herein promotesand/or increases maturation, proliferation and/or survival of germ cellsby up to 200%, up to 100%, up to 50%, up to 30%, up to 20%, up to 15%,up to 10%, or up to 5%.

In some embodiments a method, use or composition described hereinreduces, decreases or inhibits damage to germ cells in a subject. Insome embodiments a method, use or composition described herein reduces,decreases or inhibits damage to germ cells in a subject that wasadministered or delivered a chemotherapeutic agent. In some embodimentsadministration or delivery of humanin or a humanin analog reduces,decreases or inhibits damage to germ cells in a subject that wasadministered a chemotherapeutic agent. In some embodiments a method, useor composition described herein reduces, decreases or inhibits damage tothe genomic DNA, mitochondrial DNA, mitochondria, cell organelles orcell membranes of germ cells in a subject that was administered ordelivered a chemotherapeutic agent.

In some embodiments a method, use or composition described hereinenhances treatment of a hyperproliferative disorder. In some embodimentsa method, use or composition described herein enhances treatment of ametastatic or non-metastatic neoplasia, tumor, cancer or malignancy in asubject. In certain embodiments a method of enhancing treatment of ahyperproliferative disorder (e.g., a metastatic or non-metastaticneoplasia, tumor, cancer or malignancy) in a subject, comprisesadministering to the subject a composition comprising humanin and/or oneor more humanin analogs (e.g., a human polypeptide to tables 1-4). Incertain embodiments a method of enhancing treatment of ahyperproliferative disorder (e.g., a metastatic or non-metastaticneoplasia, tumor, cancer or malignancy) in a subject, comprisesadministering to the subject a composition comprising a humaninpolypeptide (e.g., humanin and/or a humanin analog) and achemotherapeutic agent. The efficacy of a treatment for ahyperproliferative disorder can be determined by any suitable method.Non-limiting examples of determining the efficacy of a treatment includedetermining the change, or lack thereof, in the number ofhyperproliferative cells and/or number of tumors, the size of tumors orthe size of a hyperproliferative tissue mass, the viability ofhyperproliferative cells, cancer progression/infiltration, for exampleby methods of cancer staging, determining the presence or extent ofmetastasis, the like or combinations thereof. Enhancing a treatmentoften comprises enhancing (e.g., improving) the efficacy of achemotherapeutic agent against a hyperproliferative disorder. In someembodiments the efficacy of a treatment that includes administration ofa chemotherapeutic agent can be enhanced or improved by administering ahumanin polypeptide prior to, concurrently with, or soon afteradministration of the chemotherapeutic agent. In some embodiments theactivity of a chemotherapeutic agent against a hyperproliferativedisorder can be increased by the presence of a humanin polypeptide.Therefore, in some embodiments the activity of a chemotherapeutic agentagainst a hyperproliferative disorder is increased by administering ahumanin polypeptide prior to, concurrently with, or soon afteradministration of the chemotherapeutic agent.

In certain embodiments, a method, use or composition herein compriseshumanin and/or a humanin analog (e.g., a humanin polypeptide). Incertain embodiments, a method or use includes administering or deliveryof humanin or a humanin analog to subject. Humanin or a humanin analogcan be administered or delivered to a subject prior to, during or afteradministration of a chemotherapeutic agent. Humanin or a humanin analogcan be administered to a subject prior to, during or after treatmentwith a chemotherapeutic agent. In certain embodiments humanin or ahumanin analog is administered or used prior to, substantiallycontemporaneously with or following administration of a chemotherapeuticagent. In certain embodiments humanin or a humanin analog isadministered or used in combination with a chemotherapeutic agent.

In some embodiments humanin or a humanin analog is administered ordelivered in an amount sufficient to protect germ cells from thecytotoxic effects of a chemotherapeutic agent. In some embodimentshumanin or a humanin analog is administered or delivered in an amountsufficient to protect germ cells in a subject from suppression or deathinduced, promoted, increased, or stimulated by a chemotherapeutic agent.In some embodiments humanin or a humanin analog is administered ordelivered in an amount sufficient to promote or increase maturation,proliferation or survival of germ cells in a subject (e.g., a subjecttreated with a chemotherapeutic agent). In some embodiments humanin or ahumanin analog is administered or delivered in an amount sufficient toincrease germ cell counts in a subject (e.g., a subject treated with anautoimmune, anti-cancer or anti-tumor chemotherapeutic agent). Methodsof determining germ cell counts are known in the art and any suitablemethod of determining germ cell counts can be used.

In some embodiments a composition comprising humanin and/or a humaninanalog is administered or delivered in an amount sufficient to inhibit achemotherapy-induced decrease or reduction of fertility. In someembodiments a composition comprising humanin and/or a humanin analog isadministered or delivered in an amount sufficient to prevent a reductionor loss of fertility due to administration of a chemotherapeutic agent.

In some embodiments humanin or a humanin analog is administered ordelivered in an amount sufficient to reduce, decrease, or inhibit damageof germ cells in a subject (e.g., a subject treated with achemotherapeutic agent).

As disclosed herein, compositions, methods and uses of the invention,can be administered or delivered prior to, contemporaneously with orafter a chemotherapeutic agent is administered or delivered, for exampleto a subject. Accordingly, methods, uses and compositions of theinvention can be delivered prior to suppression or death of germ cellsin order to protect germ cells. Compositions comprising humanin and/orone or more humanin analogs can be administered to a subjectprophylactically.

Compositions, methods and uses, such as treatment methods and uses, canprovide a detectable or measurable increase in germ cell counts,improved germ cell viability (e.g., decreased apoptosis or cell death),and/or germ cell function in a subject. Compositions, methods and usesof the invention therefore include providing a therapeutic benefit orimprovement to a subject, for example, as reflected by increased,stabilized or a less profound reduction in germ cell counts induced orpromoted by a chemotherapeutic agent, pathogenic agent and/or mechanicalactivity.

In some embodiments, a composition or method described herein enhancesan anti-cancer effect of a chemotherapeutic agent in a subject. In someembodiments, the method comprises identifying or providing a subjecthaving or suspected of having a hyperproliferative disorder andadministering a composition to the subject, wherein the compositioncomprises one or more humanin polypeptides of tables 1-4. In someembodiments, the method comprises identifying or providing a subjectthat is, has been or will receive a chemotherapy for treatment of ahyperproliferative disorder and administering a composition to thesubject, wherein the composition comprises one or more humaninpolypeptides of tables 1-4. In some embodiments, a chemotherapeuticagent is administered to a subject prior to, during or afteradministering a composition comprising a humanin polypeptide. Forexample, in certain embodiments, a chemotherapeutic agent isadministered to a subject up to 8 weeks, 1 week, 72 hr., 48 hr., 24 hr.,12 hr., 6 hr., 4 hr., 2 hr. or 1 hour before administering a compositioncomprising a humanin polypeptide. In certain embodiments, achemotherapeutic agent is administered to a subject up to 60 minutes, 45minutes, 30 minutes, 15 minutes, 10 minutes, or up to 5 minutes beforeadministering a composition comprising a humanin polypeptide. In certainembodiments, a chemotherapeutic agent is administered to a subjectduring, substantially contemporaneously with, or concurrently withadministering a composition comprising a humanin polypeptide. Achemotherapeutic agent may be administered by the same or separate routeas a composition comprising a humanin polypeptide. A compositioncomprising a chemotherapeutic agent may comprise one or more humaninpolypeptides. In some embodiments, a chemotherapeutic agent isadministered to a subject within about 72, 48, 24, 12, 6, 4, 2 or withinabout 1 hour after a composition comprising a humanin polypeptide isadministered. In some embodiments, a chemotherapeutic agent isadministered to a subject within about 60 minutes, 45 minutes, 30minutes, 15 minutes, 10 minutes, or within about 5 minutes after acomposition comprising a humanin polypeptide is administered.

In some embodiments, a composition comprising a humanin polypeptide isadministered in an amount sufficient to enhance the cytotoxic effects ofa chemotherapeutic agent on a hyperproliferative tissue. A compositioncomprising a humanin polypeptide may comprise one or more differenthumanin polypeptides. A composition comprising a humanin polypeptide maycomprise one or more humanin polypeptides of Tables 1-4. In someembodiments, a composition comprises one or more humanin polypeptidesselected from SEQ ID NO:1, SEQ ID NO:2, HNG-F6A, HN-F6A, HN-S7A andHN-C8P. In some embodiments, a composition comprises or consists of ahumanin polypeptide of SEQ ID NO:1, which composition may also include apharmaceutical acceptable carrier, salt, buffer and/or excipient. Insome embodiments, a composition comprises or consists of a humaninpolypeptide of SEQ ID NO:2, which composition may also include apharmaceutical acceptable carrier, salt, buffer and/or excipient.

A therapeutic benefit or improvement can be any measurable ordetectable, objective or subjective, transient, temporary, orlonger-term benefit to a subject or improvement in the response,disorder or disease, or one or more adverse symptoms, disorders,illnesses, pathologies, diseases, or complications caused by treatmentwith a chemotherapeutic agent (e.g., cytotoxic compound orchemotherapeutic agent) and/or associated with a disorder or disease,such as an infection Therapeutic benefits and improvements include, butare not limited to, decreasing, reducing, inhibiting, suppressing,limiting or controlling the occurrence, frequency, severity,progression, or duration of an adverse symptom of undesirable oraberrant response, disorder or disease, such as an infection.

Compositions, methods and uses of the invention, can be administered ordelivered in a sufficient or effective amount to a subject. An“effective amount” or “sufficient amount” refers to an amount thatprovides, in single or multiple doses, alone or in combination, with oneor more other compositions (e.g., chemotherapeutic agents or drugs),treatments, protocols, or therapeutic regimens, a detectable response ofany duration of time (long or short term), an expected or desiredoutcome in or a benefit to a subject of any measurable or detectabledegree or for any duration of time (e.g., for minutes, hours, days,months, years, or cured).

The doses of a “sufficient amount” for treatment (e.g., to provide atherapeutic benefit or improvement) typically are effective to provide aresponse (e.g., a measurable increase and/or stabilization) of germ cellviability, meiosis, growth, respiration, motility (e.g., sperm motility)and/or cell numbers, and/or an desired measure of fertility (e.g.,preserving fertility, preventing a loss of fertility). In someembodiments a cell viability is assayed, for example, by a reduction incell death (e.g., necrosis or apoptosis).

In some embodiments a sufficient amount humanin or a humanin analogcomprises an amount between about 0.01 to 100 mg/Kg (mg of humanin or ahumanin analog per Kg of a subjects body weight) per day, between about0.05 to 50 mg/Kg per day, between about 0.1 to 25 mg/Kg per day, betweenabout 0.5 to 15 mg/Kg per day, between about 0.5 to 15 mg/Kg per day, orbetween about 1.0 to 10 mg/Kg per day. In some embodiments administeringa sufficient amount of humanin or a humanin analog comprisesadministered one or more dose amounts of between about 0.01 to 100 mg/Kgper day, between about 0.05 to 50 mg/Kg per day, between about 0.1 to 25mg/Kg per day, between about 0.5 to 15 mg/Kg per day, between about 0.5to 15 mg/Kg per day, or between about 1.0 to 10 mg/Kg per day. Asufficient amount of humanin or a humanin analog may be administered in1, 2, 3, 4, 5, 6, or 7 doses per day. In some embodiments a sufficientamount of humanin or a humanin analog is administered continuously orintermittently by a patch or suitable device (e.g., a pump). Asufficient amount of humanin or a humanin analog may beself-administered by a subject. For example a subject may use, in one ormore doses, a sufficient amount of humanin or a humanin analog.

An effective amount or a sufficient amount can, but need not be,provided in a single administration, may require multipleadministrations, and, can but need not be, administered alone or incombination with another composition (e.g., agent), treatment, protocolor therapeutic regimen. For example, the amount may be proportionallyincreased as indicated by the need of the subject, type, status andseverity of the response, disorder, or disease treated or side effects(if any) of treatment. In addition, an effective amount or a sufficientamount need not be effective or sufficient if given in single ormultiple doses without a second composition (e.g., another drug oragent), treatment, protocol or therapeutic regimen, since additionaldoses, amounts or duration above and beyond such doses, or additionalcompositions (e.g., drugs or agents), treatments, protocols ortherapeutic regimens may be included in order to be considered effectiveor sufficient in a given subject. Amounts considered effective alsoinclude amounts that result in a reduction of the use of anothertreatment, therapeutic regimen or protocol.

As is typical for treatment methods and uses, some subjects will exhibita greater response, or less or no response to a given treatment methodor use. An effective amount or a sufficient amount therefore need not beeffective in each and every subject treated, prophylactically ortherapeutically, nor a majority of treated subjects in a given group orpopulation. An effective amount or a sufficient amount meanseffectiveness or sufficiency in a particular subject, not a group or thegeneral population. Accordingly, appropriate amounts will depend uponthe condition treated, the therapeutic effect desired, as well as theindividual subject (e.g., the bioavailability within the subject,gender, age, etc.).

Effectiveness of a method or use, such as a method of treatment hereincan provide a potential therapeutic benefit or improvement that can beascertained by various methods. Such methods include, for example,measuring germ cell counts or viability, germ cell maturation or aresponse or activity of germ cells. Measuring T or B cell activationand/or differentiation, cell infiltration of a region, cell accumulationor migration to a region, production of antibodies, cytokines,lymphokines, chemokines, interferons and interleukins, cell growth andmaturation factors using various immunological assays, such as ELISA,also can be used to ascertain effectiveness of a method, use orcomposition as set forth herein.

Humanin and/or humanin analogs, including in combinations with otheragents or drugs, can be packaged in a suitable pharmaceuticalformulation and/or dosage unit form for ease of administration anduniformity of dosage. “Dosage unit form” as used herein refers tophysically discrete units suited as unitary dosages; each unit containsa quantity of the composition optionally in association with a carrier,excipient, diluent, or vehicle calculated to produce the desiredtreatment or therapeutic (e.g., beneficial) effect. The unit dosageforms can be varied according to factors including, but not necessarilylimited to, the particular composition employed, the disorder or diseasetreated, the effect to be achieved, and the subject to be treated.Exemplary unit doses range from about 25-250, 250-500, 500-1,000,1,000-2,500, 2,500-5,000, 5,000-25,000, or 5,000-50,000 pg; from about50-500, 500-5,000, 5,000-25,000 or 25,000-50,000 ng; from about 50-500,500-5,000, 5,000-25,000 or 25,000-50,000 μg; from about 25-250, 250-500,500-1,000, 1,000-2,500, 2,500-5,000, 5,000-25,000, or 5,000-50,000 mg;and from about 1-5, 5-10, 10-25, 25-50, 50-100, 100-250, 250-500,500-1,000, 1,000-2,500, or 2,500-5,000 grams.

As set forth herein, humanin and/or humanin analogs and compositionsthereof may be contacted or provided in vitro, ex vivo or administeredor delivered in vivo to a subject or patient in various doses andamounts, and frequencies. For example, a humanin and/or humanin analogor a composition thereof can be administered or delivered to provide theintended effect, as a single or as multiple dosages, for example, in aneffective or sufficient amount.

Single or multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times)administrations or doses can be administered on the same or consecutivedays, alternating days or intermittently. For example, a humanin and/orhumanin analog or a composition thereof can be administered one, two,three, four or more times daily, on alternating days, bi-weekly, weekly,monthly, bi-monthly, or annually. A humanin and/or humanin analog orcomposition thereof can be administered for any appropriate duration,for example, for period of 1 hour, or less, e.g., 30 minutes or less, 15minutes or less, 5 minutes or less, or 1 minute, or less.

A humanin and/or humanin analog or composition thereof can beadministered to a subject and methods and uses may be practiced priorto, substantially contemporaneously with, or within about 1-60 minutes,hours (e.g., within 1, 2, 3, 4, 5, 6, 8, 12, 24 hours), or days (1, 2,3, 4, 5, 6, 7, 7-14, 14-21, 21-28, 28-45, 45-60, 60-90, etc.) ofadministration of a chemotherapeutic agent.

A humanin and/or humanin analog or composition thereof can beadministered or delivered and methods and uses may be practiced viasystemic, regional or local administration, by any route. For example, ahumanin and/or humanin analog or composition thereof may be administeredor delivered systemically, regionally or locally, via injection,infusion, orally (e.g., ingestion or inhalation), topically,intravenously, intra-arterially, intramuscularly, intraperitoneally,intradermally, subcutaneously, intracavity, intracranially,transdermally (topical), parenterally, e.g. transmucosally orintrarectally (enema) catheter, or optically. Humanin and/or humaninanalog, compositions, methods and uses of the invention includingpharmaceutical formulations may be administered via a(micro)encapsulated delivery system or packaged into an implant foradministration.

As used herein the term “pharmaceutically acceptable” and“physiologically acceptable” mean a biologically acceptable formulation,gaseous, liquid or solid, or mixture thereof, which is suitable for oneor more routes of administration, in vivo delivery or contact. A“pharmaceutically acceptable” or “physiologically acceptable”composition is a material that is not biologically or otherwiseundesirable, e.g., the material may be administered to a subject withoutcausing substantial undesirable biological effects. Thus, such apharmaceutical composition may be used, for example in a formulation foradministering or delivering a humanin and/or humanin analog orcomposition thereof to a subject.

Such compositions include solvents (aqueous or non aqueous), solutions(aqueous or non aqueous), emulsions (e.g., oil-in-water orwater-in-oil), suspensions, syrups, elixirs, dispersion and suspensionmedia, coatings, isotonic and absorption promoting or delaying agents,compatible with pharmaceutical administration or in vivo contact ordelivery. Aqueous and non-aqueous solvents, solutions and suspensionsmay include suspending agents and thickening agents. Suchpharmaceutically acceptable carriers include tablets (coated oruncoated), capsules (hard or soft), microbeads, powder, granules andcrystals. Supplementary active compounds (e.g., preservatives,antibacterial, antiviral and antifungal agents) can also be incorporatedinto the compositions.

Pharmaceutical compositions can be formulated to be compatible with aparticular route of administration or delivery, as set forth herein orknown to one of skill in the art. Thus, pharmaceutical compositionsinclude carriers, diluents, or excipients suitable for administration byvarious routes.

Compositions suitable for parenteral administration comprise aqueous andnon-aqueous solutions, suspensions or emulsions of the active compound,which preparations are typically sterile and can be isotonic with theblood of the intended recipient. Non-limiting illustrative examplesinclude water, saline, dextrose, fructose, ethanol, animal, vegetable orsynthetic oils.

For transmucosal or transdermal administration (e.g., topical contact),penetrants can be included in the pharmaceutical composition. Penetrantsare known in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.For transdermal administration, the active ingredient can be formulatedinto aerosols, sprays, ointments, salves, gels, or creams as generallyknown in the art. For contact with skin, pharmaceutical compositionstypically include ointments, creams, lotions, pastes, gels, sprays,aerosols, or oils. Carriers which may be used include petroleum jelly,lanolin, polyethylene glycols, alcohols, transdermal enhancers, andcombinations thereof.

Cosolvents and adjuvants may be added to the formulation. Non-limitingexamples of cosolvents contain hydroxyl groups or other polar groups,for example, alcohols, such as isopropyl alcohol; glycols, such aspropylene glycol, polyethyleneglycol, polypropylene glycol, glycolether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acidesters. Adjuvants include, for example, surfactants such as, soyalecithin and oleic acid; sorbitan esters such as sorbitan trioleate; andpolyvinylpyrrolidone.

Appropriate pharmaceutical compositions and delivery systems are knownin the art (see, e.g., Remington: The Science and Practice of Pharmacy(2003) 20th ed., Mack Publishing Co., Easton, Pa.; Remington'sPharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton,Pa.; The Merck Index (1996) 12th ed., Merck Publishing Group,Whitehouse, N.J.; Pharmaceutical Principles of Solid Dosage Forms(1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel andStoklosa, Pharmaceutical Calculations (2001) 11th ed., LippincottWilliams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug DeliverySystems (1980), R. L. Juliano, ed., Oxford, N.Y., pp. 253-315).

The invention provides kits comprising humanin and/or humanin analogs,combination compositions and pharmaceutical formulations thereof,packaged into suitable packaging material. A kit optionally includes alabel or packaging insert including a description of the components orinstructions for use in vitro, in vivo, or ex vivo, of the componentstherein. Exemplary instructions include instructions for a method,treatment protocol or therapeutic regimen. The term “packaging material”refers to a physical structure housing the components of the kit. Thepackaging material can maintain the components sterilely, and can bemade of material commonly used for such purposes (e.g., paper,corrugated fiber, glass, plastic, foil, ampules, vials, tubes, etc.).

Kits can include labels or inserts. Labels or inserts include “printedmatter,” e.g., paper or cardboard, or separate or affixed to acomponent, a kit or packing material (e.g., a box), or attached to anampule, tube or vial containing a kit component. Labels or inserts canadditionally include a computer readable medium, optical disk such asCD- or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storagemedia such as RAM and ROM or hybrids of these such as magnetic/opticalstorage media, FLASH media or memory type cards.

Labels or inserts can include identifying information of one or morecomponents therein, dose amounts, clinical pharmacology of the activeingredient(s) including mechanism of action, pharmacokinetics (PK) andpharmacodynamics (PD). Labels or inserts can include informationidentifying manufacturer information, lot numbers, manufacturer locationand date.

Labels or inserts can include information on a condition, disorder,disease or symptom for which a kit component may be used. Labels orinserts can include instructions for the clinician or for a subject forusing one or more of the kit components in a method, treatment protocolor therapeutic regimen. Instructions can include dosage amounts,frequency or duration, and instructions for practicing any of themethods or uses, treatment protocols or therapeutic regimes set forthherein. Kits of the invention therefore can additionally include labelsor instructions for practicing any of the methods and uses of theinvention described herein.

Labels or inserts can include information on any benefit that acomponent may provide, such as a prophylactic or therapeutic benefit.Labels or inserts can include information on potential adverse sideeffects, such as warnings to the subject or clinician regardingsituations where it would not be appropriate to use a particularcomposition. Adverse side effects could also occur when the subject has,will be or is currently taking one or more other medications that may beincompatible with the composition, or the subject has, will be or iscurrently undergoing another treatment protocol or therapeutic regimenwhich would be incompatible with the composition and, therefore,instructions could include information regarding such incompatibilities.

Kits can additionally include other components. Each component of thekit can be enclosed within an individual container and all of thevarious containers can be within a single package. Invention kits can bedesigned for cold storage.

EXAMPLES

The examples set forth below illustrate certain embodiments and do notlimit the technology.

Example 1 Experiments 1-4, HN, HN Analogs and Effects of HN and Analogson Germ Cell Apoptosis Induced by Chemotherapeutic Drugs

Humanin (HN), a 24-amino acid mitochondrial derived peptide, is anendogenous anti-apoptotic peptide in many tissues including the testis.Experiments were performed herein to explore the application of HN andits analogs as cytoprotective agents for protecting germ cells fromchemotherapy-induced apoptosis (e.g., for fertility preservation).Studies were also performed to determine the ability of HN and itsanalogs to alter the effects of chemotherapeutic agents against cancercells.

As disclosed herein, it was determined that HN has cytoprotectiveeffects on two chemotherapeutic agents in two models: doxorubicin (DOX)to induce germ cell apoptosis in ex vivo seminiferous tubule cultures(Experiment 1) and CP in vivo treatment in adult mice (Experiment 2).Accordingly, HN is an effective protector of germ cell death in micetreated with chemotherapy and thus is a protector against chemotherapyinduced infertility.

A potent humanin analog HNG was used with or without chemotherapeuticagent CP in a mouse melanoma lung metastasis model (Collisson, et al.,2003; Craft, et al., 2005) (Experiment 3 and 4). The synthetic HNanalog, HNG, has a substitution of glycine for serine at position 14(HNG-S14G) in the 24 amino acid sequence of HN, which enhances itscytoprotective activity (Hashimoto, et al., 2001).

Moreover, HN and its analogs HNG can be an effective adjunct toestablished chemotherapy to markedly enhance the anti-cancer effects ofchemotherapy treatment of a metastatic melanoma mouse model. Thesediscoveries indicate clinical relevance to markedly enhance treatment ofcancer and at the same time protect the cured patient against treatmentinduced infertility.

Whether HN would prevent chemotherapy-induced germ cell apoptosis wasinvestigated. Segments of seminiferous tubules isolated from adult mousetestis ex vivo were treated with either HN, doxorubicin (DOX) or HN+DOXfor 12 hours. Young adult mice in vivo were treated with intraperitoneal(IP) injection of HN, cyclophosphamide (CP), or the combination. Humaninsignificantly reduced germ cell apoptosis in mice induced by DOX inseminiferous tubule cultures ex vivo and by CP in mice in vivo (bothp<0.05). These data showed that HN reduces chemotherapy-induced germcell apoptosis in mice. In some embodiments, synthetic HN or its analogsmay reduce chemotherapy-induced cancer cell death while protecting germcells from apoptosis. To verify this, a potent HN analog HNG (HN-S14G)was used with or without CP in a mouse melanoma with lung metastasismodel. Five mice were used as control and 20 mice were inoculatedintravenously with B16 murine melanoma cells. Of these mice fivereceived no further treatment. A week later the remaining 15 mice (n=5per group) were treated for additional 2 weeks with: 1) HNG daily IPinjection for 2 weeks; 2) a single CP IP injection; 3) combined CP withHNG. All mice were sacrificed 3 weeks after tumor inoculation.Non-treated tumor-bearing mice had increased germ cells apoptosis whencompared with control (p=0.001). CP treatment significantly increasedgerm cell apoptosis (P<0.001) in comparison with control and HNG treatedmice. HNG treatment for 2 weeks attenuated CP induced germ cellapoptosis (p<0.001). While HNG treatment decreased the number ofmetastatic tumors in the lungs, CP treatment not only decreased numberof tumors (p=0.006) but also regressed tumor size when compared withnon-treated tumor-bearing mice. Importantly, combined HNG and CPtreatment significantly decreased number of tumors (p<0.001) and led tomore to differentiated melanin producing cells compared to CP treatmentalone. This indicates that 1) HNG protects male germ cells fromapoptosis induced by CP treatment; 2) HNG enhances the suppressiveeffects of CP on metastatic lung melanoma in mice and markedly increasenumber of differentiated melanoma cells compared to CP alone. The longterm effects and the mechanisms of action of HNG on male fertilitypreservation and the regression of cancer progression will be determinedin future planned experiments.

Materials and Methods Animals

Adult male C57BL/6J (25 to 30 g) mice were obtained from JacksonLaboratory (Bar Harbor, Me.) and housed at the accredited animalfacilities at Los Angeles Biomedical Research Institute. The mice hadunlimited access to food and water and were provided housing at normallight-dark cycles (12 hr. each) at a constant temperature of 22° C.Animal handling, experimentation, and killing of the animals were inaccordance with the recommendation of the American Veterinary MedicalAssociation and were approved by the Animal Care and Use ReviewCommittee of Los Angeles Biomedical Research Institute at Harbor-UCLAMedical Center.

Materials

HN and HNG were synthesized by CPC Scientific (Sunnyvale, Calif.), andDOX (doxorubicin hydrochloride) and CP (cyclophosphamide monohydrate)were obtained from Sigma Aldrich (St. Louis, Mo.).

Experimental Design and Procedures Experiment 1 The Effects of HN onDOX-Induced Germ Cell Apoptosis in Mouse Seminiferous Tubule Cultures ExVivo

A total of 15 young adult (12-13 week-old) mice were used for these exvivo studies. The animals were sacrificed with a 5% pentobarbital (200mg/Kg) IP injection. Testes were dissected and after removing the tunicaalbuginea, seminiferous tubules isolated from testes were cut into smallsegments under a dissecting microscope and light and dark segments wereseparated by dissection. Ten to twelve segments were placed (2 mm inlength) in each well on a six well plate with 2 ml serum free Ham F-10culture medium (GIBCO, Life Technologies, Thermo Fisher ScientificCorp., Carlsbad, Calif.) with the following treatments: control (Con,n=10), heat (43° C., 15 minutes, used as a positive control, n=10); HN10 μg/mL (n=9); DOX 10 mcM (Dox10, n=10); and DOX 10 mcM+Humanin 10μg/mL (Dox10+HN, n=10). For these experiments, n represents the totalnumber of times the treatment was repeated using different animals.After 12 hours of incubation at 34° C. the seminiferous tubules fromeach treatment group were used to make “squashed” seminiferous tubulesamples on a slide for TUNEL (TdT-mediated dUTP nick-end labeling) assayto detect germ cell apoptosis (Erkkila, et al., 1997; Toppari &Parvinen, 1985). To quantify the rate of germ cell apoptosis under alight microscope at 400× magnification, a square grid fitted within theeyepiece provided a reference area of 62,500 μm². The TUNEL positiveapoptotic germ cells within the frame of grid were counted in 4 segmentsof seminiferous tubules in each group. The incidence of germ cellapoptosis was expressed as the number of apoptotic germ cells per mm².

Experiment 2 The In Vivo Effects of HN on CP Induced Male Germ CellApoptosis in Mice

Young adult mice (12-13 weeks) were randomly divided into 4 groups with4 or 5 mice per group and received an IP injection: control (Con)(saline), HN (40 mg/Kg BW), CP (200 mg/Kg BW), and the combination ofCP+HN. These experiments were repeated four times. In the CP+HN group,mice received an injection of HN followed by an injection of CP about 15minutes later. Twenty-four hours after injections animals weresacrificed using 5% pentobarbital (200 mg/kg BW) IP injection. Blood wascollected and one testis was removed, weighed and frozen in liquidnitrogen. The mouse was next perfused with saline and then subsequentlyperfuse-fixed with Bouin's solution as previously described andapoptosis was assessed by TUNEL assay (Lue, et al., 2009).

Experiment 3 Effect of HNG on CP-Induced Germ Cell Apoptosis andMetastatic Lung Tumor Regression in a Mouse B16 Melanoma Model

A total of 25 young adult mice (10-12 weeks) were randomly divided into5 groups (n=5 per group). Five mice were used as control. Twenty micewere inoculated intravenously with B16 murine melanoma cells (2×10⁵cells/mouse). Among these 20 tumor-inoculated mice, 5 mice received nofurther treatment. After one week, the remaining 15 mice were dividedinto 3 groups and injected with: 1) HNG daily IP (5 mg/Kg body weight)for 2 weeks; 2) CP (200 mg/Kg body weight IP single injection); 3)combined CP with HNG treatment (same doses as above). All mice weresacrificed 3 weeks after tumor inoculation, and 2 weeks after HNG and/orCP treatment. The animals were sacrificed with 5% pentobarbital (200mg/kg body weight) IP injection after which blood was collected from theright atrium of the heart. One of the testes from each mouse wasimmersed fixed in Bouin's solution, and the mice were perfused withsaline and subsequently perfuse-fixed with 5% glutaraldehyde in 0.05 Mcacodylate buffer (pH 7.4). The lungs were dissected out and post fixedin 5% glutaraldehyde solution for 24 hours and stored in cacodylatebuffer at 4° C. for the subsequent tumor count (Lue, et al., 2010; Lue,et al., 2009).

Experiment 4 Effect of Higher Doses of HNG on CP-Induced Germ Cell Deathand Enhancement of CP in Suppressing Tumor Growth

Experiment 3 was repeated after inoculation of B16 melanoma tumor atweek 1 and then treated with the following treatment starting at week 2(n=5 mice per group): 1). No treatment; 2). HNG IP injections 5 mg/Kgdaily; 3) CP 200 mg/Kg single IP injection; 4) HNG 5 mg/Kg/day+CP; 5).HNG IP injections 15 mg/Kg daily; and 6). HNG 15 mg/Kg/day+CP. Allanimals were euthanized and perfused with 4% paraformaldehyde at the endof week 3. Lungs and testes were collected and processed as inexperiment 3.

Methods

For detection of apoptosis in testicular sections, the paraffin embeddedtestis tissue was cut into 5 micron sections and stained with theTdT-mediated dUTP nick-end labeling assay (TUNEL Apoptosis DetectionKit, Millipore, Billerica, Mass.) to detect apoptosis of germ cells inthe testis (Lue, et al., 2009). Testicular sections assayed by TUNELwere examined under a light microscope and the apoptotic germ cells wereblindly and manually counted. The seminiferous tubules in the testissection under the microscope were grouped as stages IX-IV (early-latestages of spermatogenesis) and V-VIII (middle stages ofspermatogenesis). Germ cell apoptosis was quantified as the number ofseminiferous tubules containing apoptotic spermatogonia, spermatoctyes,round spermatids and spermatozoa per 100 seminiferous tubules (Drumond,et al., 2011).

B16 melanoma tumors in the lung were quantified by counting the numberof tumors on the surface of the lungs under stereomicroscopy (Collisson,et al., 2003; Craft, et al., 2005). The size of the tumor was measuredusing histomorphometry and expressed as average size of tumor. Theappearance of the melanoma cells were reviewed and characterized by anexpert pathologist.

Statistical Analysis

Statistical analyses of the majority of the data was performed using theSigma Plot 12 program (Jandel, San Rafael, Calif.). The data wereanalyzed by using t-tests and one-way ANOVA followed byStudent-Newman-Keuls Method. P-values of less than 0.05 (P<0.05) wereconsidered significant. In in vivo experiments when the testicularsections were assessed for apoptosis, a mixed model used the percentageof tubules with apoptosis as the dependent variable. Two differentstages (Early or Late Vs Middle) and four treatment groups (Control, HNCP, CP+HN) as well as the interaction term were included in the model asindependent variables. Persons who did the experiment were considered asa random effect to accommodate a potential heterogeneous variance in themodel. Least Square Means and their differences were computed and testedusing the Tukey's adjustment for multiple pair-wise comparisons. Aresidual analysis was done to check for the model assumptions. All thedata analysis was carried out using SAS 9.3 (Cary, N.C. USA).

Results Experiment 1 Results Effect of Doxorubicin on Germ CellApoptosis in Ex Vivo Seminiferous Tubule Cultures

It was indicated in short term (12 hours) ex vivo seminiferous tubulecultures that HN (10 μg/mL) treatment alone had no effect on germ cellapoptosis when compared with control. As expected, heat exposure servingas a positive control (43° C., 15 minutes), significantly increased therate of apoptosis compare to control and HN treated groups (p<0.05).Addition of DOX at 10 micromole induced a significantly higher number ofapoptotic germ cells when compared to control and HN treated groups(p<0.05) (FIG. 1). Treatment with HN (10 μg/ml) significantly reducedthe number of apoptotic germ cells induced by DOX treatment (p<0.05)(FIG. 1).

Experiment 2 Results Effect of HN on CP-Induced Germ Cell Apoptosis InVitro

In experiment 2, the body and testis weights of mice from the 4experimental groups were not significantly different 24 hours aftertreatment (FIG. 2). As shown in the testicular cross sections stainedwith TUNEL (FIG. 3), compared to the control group (FIG. 3A), HN (40mg/Kg, IP single injection) alone (FIG. 3B) had no significant effect ongerm cell apoptosis. CP treatment (FIG. 3C) significantly increased germcell apoptosis. HN treatment attenuated the CP-induced germ cellapoptosis (FIG. 3D). Examination of the effect of HN on stage-specificgerm cell apoptosis showed that in early-late stages of spermatogenesis(Stages XI-IV)(FIG. 4), CP treatment significantly increased germ cellapoptosis compared to control (p=0.003) and HN (p-0.003)(FIG. 4). TheCP-induced apoptosis was ameliorated by concomitant treatment with HN(CP+HN, p<0.033) but not to the extent of apoptosis observed in control(p=0.052) and HN (p=0.046) treated mice (FIG. 4E). In the middle stages(V-VIII, FIG. 5) stages of spermatogenesis CP induced significantincrease in apoptosis compared to control (p=0.002) and HN (p=0.003)groups. The CP-induced apoptosis in the middle stages was notsignificantly decreased by concomitant treatment with HN (CP+HN, p=0.29)and CP+HN remained higher than control (p=0.006) and HN (p=0.008)treated mice (FIG. 5E).

Experiment 3 Results Effect of HNG on CP Induced Germ Cell Apoptosis andMetastatic Lung Tumor Regression in a Mouse B16 Melanoma Model

The mean body weight was similar in the five groups of mice before B16mouse melanoma cell injection into the tail vein (FIG. 6A). Three weeksafter tumor inoculation, mice that were not treated had significantlylower body weight when compared with control, HNG alone, CP alone andHNG+CP treated mice (FIG. 6B). CP treatment for 2 weeks significantlydecreased the testis weight in comparison with control, non-treatedtumor bearing, and HNG treated mice (FIG. 7). Addition of HNG to CPtreatment failed to restore the testis weight to the control,non-treated, and HN treated groups (FIG. 7). Non-treated mice withmetastatic melanoma had increased germ cells apoptosis when comparedwith control (FIG. 8). CP treatment further significantly increased germcell apoptosis (P<0.001) when compared to control and HNG treated mice.Daily HNG treatment for 2 weeks significantly (p<0.001) attenuatedCP-induced germ cell apoptosis (FIG. 8).

The lungs were examined and the number of metastatic tumors on thesurface of the lungs was quantified (FIG. 9). HNG treatment aloneappeared to decrease the number of tumors but appeared to have no effecton the size of the melanoma metastasis on the surface of the lungs. Onthe other hand, CP treatment not only significantly (p=0.006) decreasednumber of tumors but also tumor size as compared with non-treatedtumor-bearing mice (FIG. 9, row CP and FIG. 10). Importantly, additionof HNG to CP treatment significantly (p<0.001) (FIG. 9. row CP+HNG andFIG. 10) decreased the number of tumors as compared to CP treatmentalone (FIG. 9, row CP). Tumor size was assessed by histomorphometry.

Experiment 4

Using the same methods as Experiment 3, the number of tumors on thesurface of lungs of mice treated with saline as non-treated control werecounted, CP alone, HNG (5 mg/Kg BW), HNG (5 mg/Kg BW)+CP, HNG (15 mg/KgBW), and HNG (15 mg/kg BW)+CP. All animals were sacrificed 3 weeks afterB16 Cell Injection, and 2 weeks after HNG, CP or CP+HNG treatment. Asdemonstrated in FIG. 11, HNG at both 5 mg/kg BW (p=0.0043) and 15 mg/kgBW (p=0.0029) doses significantly decreased the number of lung tumorsbut not size of the tumors when compared with non-treated tumor-bearingmice. CP significantly decreased both the number and size of the tumorscompared to controls (p=0.013). HNG at both 5 mg/Kg BW and 15 mg/Kg BWwhen added concomitantly with CP significantly decreased the number oftumors than either treatment alone (P=0.001). The assessment of lungtumor area and testicular germ cell apoptosis is being assessed in thelaboratory using histomorphometry. Histological examination of themetastatic lung tumor cells showed that CP decreased the number ofmetastatic melanoma cells, whereas HNG was added to the CP treatmentresulted in much more differentiation of the metastatic lung tumor cellswhen compared to CP treatment alone (FIG. 12).

Discussion

Chemotherapeutic agents in combination can be very effective intreatment of leukemia, lymphoma, testicular cancers, brain tumors andmelanoma. These cancers occur commonly in young men. Preservation offertility in young patients with cancer after chemotherapy is important.DOX and CP or other anticancer drugs with similar mechanisms of actionare currently used in combined chemotherapy regimens for cancerpatients. Both DOX and CP administration induces cancer cell apoptosisbut also damages testicular germ cells in both animal models and men(Dohle, 2010; Marcon, et al., 2011; Meistrich, 2013; Meistrich, et al.,1982; Trost & Brannigan, 2012). Prior studies indicate that HN candecrease male germ cell apoptosis induced by intratesticular hormonaldeprivation (Jia, et al., 2013; Lue, et al., 2010).

The current study showed that DOX which has activity in vitro inducedgerm cell apoptosis in male germ cells which was attenuated when DOX wasadministered with HN. In addition, in vivo HN treatment reduced thecytotoxic effects of CP in the testis in mice. Results indicated that HNprevents CP-induced apoptosis mainly in the early and late stages of theseminiferous epithelium, as spermatogenesis progresses, germ cell fromall stages will be protected by HN treatment as seen in experiment 3.Methods disclosed herein provide HN as a means of cytoprotective effectof on male germ cells after cancer chemotherapy. Synthetic HNadministration may also reduce testicular stress induced germ cellapoptosis through interactions of endogenous HN with BAX in thecytoplasm preventing the entry of BAX to the mitochondria to prevent theinitiation of the apoptotic cascade in the testis (Jia, et al., 2013).

HN's protective effect on male germ cells from CP-induced apoptosis in amouse melanoma with lung metastasis model was studied. Non-treatedtumor-bearing mice had increased germ cell apoptosis compared tocontrols (no melanoma), indicating lung metastases induced weight lossand poor health causing defective spermatogenesis. HNG treatment showeda trend to mitigate germ cell apoptosis in tumor-bearing mice. CPtreatment mainly induces differentiated spermatogonia and spermatocytesapoptosis in mice (Drumond et al., 2011). Consistent with publisheddata, most apoptotic spermatocytes were cleared by phagocytosis bySertoli cells 2 weeks after CP treatment. The increased percentage oftubules with apoptotic germ cells 2 weeks after CP treatment indicate aprolonged effect of CP on germ cells or impaired clearance of dead cellsby Sertoli cells in tumor-bearing mice. However, HNG induced decrease ingerm cell apoptosis failed to prevent testis weight loss induced by CPtreatment in 2 weeks. Thus, long term study is needed to investigate theeffects of HN and its analog on fertility preservation, blockingspermatozoa DNA damage, and possible minimizing the adverse effects onthe progeny in response to chemotherapy.

HN gene expression has been found abundantly in lymphoma and gastriccancer indicating that HN may be an oncopeptide protecting cancer cellfrom undergoing apoptosis (Hartmann, et al., 2008; Maximov, et al.,2002; Mottaghi-Dastjerdi, et al., 2014; Tarantul & Hunsmann, 2001). Inthe same mouse melanoma model the effect of HNG on tumor growth andprogression with or without concomitant CP treatment was screened. Itwas indicated that HNG administration alone for 2 weeks moderatelydecreased the number of metastatic lung melanomas without affecting thesize of the lung tumors. HNG enhances the anti-cancer effects of CP onmetastatic lung melanoma formation in mice.

These data provide evidence that HNG may have opposing actions on normalversus cancer cells: HNG protects normal cells from stress-inducedapoptosis and enhances cancer regression in response to chemotherapy inmice. Because of the large intra-group variations on number of tumorsfound on the surface after HNG treatment alone, the HNG effect on tumorgrowth and apoptosis has to be determined in a larger scale experimentswith different doses of HNG administration. Furthermore, the mechanismsof the effect of HNG in conjunction with CP treatment on the suppressionof metastatic lung melanoma formation remain to be determined. Itappears that HNG may either has direct inhibitory action on metastaticmelanoma proliferation or promote melanoma cell differentiation in thelung or indirect action by modulating the microenvironment of themetastatic lung melanoma such as decreasing angiogenesis or alteringimmune cells homing to the cancer. CP and HNG may activate differentsignaling pathways in melanoma cells which resulted in enhancedanti-tumor effects. CP has a direct action on DNA causing DNA breaks,HNG may accelerate tumor cell differentiation or death throughmitochondrial pathways. Another unexplored mechanism is the effect ofHNG on the host defense mechanisms against the invasion of the lungs bymetastatic melanoma.

In summary, 1) HN and HNG protected germ cell from apoptosis induced byDOX and CP treatment; 2) HNG enhanced the anti-cancer effect on CPsuppression of lung metastatic melanoma formation in mice (includingdecreasing the number and converting undifferentiated cancer todifferentiated cancer cells). Differentiation of otherwise highlymitotic undifferentiated metastatic cells may imply conversion to lessaggressive malignancy. The combination of greater reduction of number ofmetastatic cancer cells and the reversion to more differentiatedresidual cells by combined HNG and chemotherapy, predicts that thiscombined therapy will be advantageous over chemotherapy alone. Theeffects of HNG to reduce apoptosis of germ cells when treated withchemotherapy indicated that this combined therapy will reduce andperhaps prevent the infertility seen by chemotherapy alone.

Humanin (HN) Polypeptides and Analogs (Tables 1-4)

TABLE 1 Table 1: HN polypeptides - Properties and Cytoprotective ActionHN Mutant Mutation Characteristics Cytoprotective Action HN-F6A Phe6 toAla Loss of IGFBP-3 binding Similar/more effective than HN. HN-S7A Ser7to Ala Loss of membrane receptor Not effective, prevents HN self- or HN-Cys8 to Ala binding dimerization C8A HN-C8P Cys8 to Pro Loss of BAXbinding Not effective, blocks intracellular HN action HN-L12A Leu12 toDimerizes with and HN antagonist, forms inactive dimer Ala inactivatesHN with HN HN-S14G Ser14 to Same mechanisms of 10 to 1000 times morepotent than HN Gly action as HN in some cells.

TABLE 2 HN Polypeptides and Analogs SEQ ID Name Amino acid sequence NOHumanin MAPRGFSCLLLLTSEIDLPVKRRA SEQ ID (HN) NO: 1 S14G-HNMAPRGFSCLLLLTGEIDLPVKRRA SEQ ID (HNG) NO: 2 D-Ser14MAPRGFSCLLLLT(DS)EIDLPVKRRA SEQ ID HN NO: 3 AGA-HNGMAPAGASCLLLLTGEIDLPVKRRA SEQ ID NO: 4 AGA-(D-MAPAGASCLLLLT(DS)EIDLPVKRRA SEQ ID Ser14)HN NO: 5 AGA-(D-PAGASCLLLLT(DS)EIDLP SEQ ID Ser14) NO: 6 HN17 AGA-(C8R)PAGASRLLLLTGEIDLP SEQ ID HNG17 NO: 7 EF-HNEFLIVIKSMAPRGFSCLLLLTSEIDLPVKRRA SEQ ID NO: 8 EF-HNGEFLIVIKSMAPRGFSCLLLLTGEIDLPVKRRA SEQ ID NO: 9 EF-AGA-EFLIVIKSMAPAGASCLLLLTGEIDLPVKRRA SEQ ID HNG NO: 10 ColivelinSALLRSIPAPAGASRLLLLTGEIDLP SEQ ID NO: 11 L9R-HN MAPRGFSCRLLLTSEIDLPVKRRASEQ ID NO: 12 Humania MTPRGFSCLLLPTSETDLPVKRRX SEQ ID (7) NO: 13 HumaniaMAPRGFSCLLLSTSEIDLPVKRXX SEQ ID (5) NO: 14 HumaniaMAPRGFSCLLLSTSEIDLPVKRRA SEQ ID (3/11) NO: 15 SHLP1MCHWAGGASNTGDARGDVFGKQAG SEQ ID NO: 16 SHLP2 MGVKFFTLSTRFFPSVQRAVPLWTNSSEQ ID NO: 17 SHLP3 MLGYNFSSEPCGTISIAPGFNFYRLYFIWV SEQ ID NGLAKVVWNO: 18 SHLP4 MLEVMFLVNRRGKICRVPF1FFNLSL SEQ ID NO: 19 SHLPSMYCSEVGFCSEVAPTEIFNAGLVV SEQ ID NO: 20 SHLP6 MLDQDIPMVQPLIKVRLFND SEQ IDNO: 21 HN-F6A MAPRGASCLLLLTSEIDLPVKRRA SEQ ID NO: 22 HNG-F6AMAPRGASCLLLLTGEIDLPVKRRA SEQ ID NO: 23 HN-S7A MAPRGFACLLLLTSEIDLPVKRRASEQ ID NO: 24 HN-C8P MAPRGFSPLLLLTSEIDLPVKRRA SEQ ID NO: 25

TABLE 3 HN Polypeptides and Analogs P-S14 HN 4MAPRGFSCLLLLT(p-S)EIDLPVKRRA SEQ ID NO: 26 P-S7 HN 5MAPRGF(p-S)CLLLLTSEIDLPVKRRA SEQ ID NO: 27 P-S7/14 HN 6MAPRGF(p-S)CLLLLT(p-S)EIDLPVKRRA SEQ ID NO: 28 D-Ser14 HN 7MAPRGFSCLLLLT(D-S)EIDLPVKRRA SEQ ID NO: 29 D-Ser7 HN 8MAPRGF(D-Ser)CLLLLTSEIDLPVKRRA SEQ ID NO: 30 D-Ser7/14MAPRGF(D-Ser)CLLLLT(D-Ser)EIDLPVKRRA HN 9 SEQ ID NO: 31 AGA-(D-Ser14)MAPAGASCLLLLT(D-Ser)EIDLPVKRRA HN 10 SEQ ID NO: 32 AGA-(D-Ser14)PAGASCLLLLT(D-Ser)EIDLP HN17 11 SEQ ID NO: 33 EF-(S7A)HN 15EFLIVIKSMAPRGFACLLLLTSEIDLPVKRRA SEQ ID NO: 34 EF-HNG-KKK 16EFLIVIKSMAPRGFSCLLLLTGEIDLPVKKKK SEQ ID NO: 35 EF-HN 17EFLIVIKSMAPRGFSCLLLLTSEIDLPVKRRA SEQ ID NO: 36 EH-HNA 18EFLIVIKSMAPRGFSALLLLTSEIDLPVKRRA SEQ ID NO: 37 EF-HNG 19EFLIVIKSMAPRGFSCLLLLTGEIDLPVKRRA SEQ ID NO: 38 EF-AGA-HNG 22EFLIVIKSMAPAGASCLLLLTGEIDLPVKRRA SEQ ID NO: 39

TABLE 4 HN Polypeptides and Analogs HN 1 MAPRGFSCLLLLTSEIDLPVKRRASEQ ID NO: 40 HNG 2 MAPRGFSCLLLLTGEIDLPVKRRA SEQ ID NO: 41 HNA 3MAPRGFSALLLLTSEIDLPVKRRA SEQ ID NO: 42 P-S14 HN 4MAPRGFSCLLLLT(p-S)EIDLPVKRRA SEQ ID NO: 43 P-S7 HN 5MAPRGF(p-S)CLLLLTSEIDLPVKRRA SEQ ID NO: 44 P-57/14 HN 6MAPRGF(p-S)CLLLLT(p-S)EIDLPVKRRA SEQ ID NO: 45 D-Ser14 HN 7MAPRGFSCLLLLT(D-S)EIDLPVKRRA SEQ ID NO: 46 D-Ser7 HN 8MAPRGF(D-Ser)CLLLLTSEIDLPVKRRA SEQ ID NO: 47 D-Ser7/14MAPRGF(D-Ser)CLLLLT(D-Ser)EIDPPVKRRA HN 9 SEQ ID NO: 48 AGA-(D-Ser14)MAPAGASCLLLLT(D-Ser)EIDLPVKRRA HN 10 SEQ ID NO: 49 AGA-(D-Ser14)PAGASCLLLLT(D-Ser)EIDLP HN17 11 SEQ ID NO: 50 S7A HN 12MAPRGFACLLLLTSEIDLPVKRRA SEQ ID NO: 51 S7A HNG17 13 PRGFACLLLLTSEIDLPSEQ ID NO: 52 HNG-KKK 14 YMAPRGFSCLLLLTGEIDLPVKKKK SEQ ID NO: 53EF-(S7A)HN 15 EFLIVIKSMAPRGFACLLLLTSEIDLPVKRRA SEQ ID NO: 54EF-HNG-KKK 16 EFLIVIKSMAPRGFSCLLLLTGEIDLPVKKKK SEQ ID NO: 55 EF-HN 17EFLIVIKSMAPRGFSCLLLLTSEIDLPVKRRA SEQ ID NO: 56 EH-HNA 18EFLIVIKSMAPRGFSALLLLTSEIDLPVKRRA SEQ ID NO: 57 EF-HNG 19EFLIVIKSMAPRGFSCLLLLTGEIDLPVKRRA SEQ ID NO: 58 EFLIVIKS 20 EFLIVIKSSEQ ID NO: 59 AGA-HNG 21 MAPAGASCLLLLTGEIDLPVKRRA SEQ ID NO: 60EF-AGA-HNG 22 EFLIVIKSMAPAGASCLLLLTGEIDLPVKRRA SEQ ID NO: 61 HNG-17 23PRGFSCLLLLTGEIDLP SEQ ID NO: 62

HNG: An HN derivative, which has a Gly substitution of Ser14 of HN.

HN derivatives can be selected from: Humanin with S14P, P-S7 HN, P-S7/14HN, (D-Ser14)HN, (D-Ser7)HN, (D-Ser7/14)HN, AGA-(D-Ser14)HN, AGA-(D-Seri4)HN17, EFLIVIKS-HNG, EFLIVIKS-HNA, EFLIVIKS-HN, EFLIVIKS-HNG-KKK,EFLIVIKS-(S7A)HN, and EFLIVIKS-AGA-HNG, and chimeric combinationsthereof. The “514P” means that the S (serine) at location 14 in thewild-type HN has been replaced with P (proline). The same conventionapplies for other substitutions (e.g., S7A). “D-Ser7” means that theSerine at location 7 has been switched (racemized) from a normalL-isomer to the D-isomer. “AGA-HN” is a shorthand name of the HNderivative in which the Arg4 and Phe6 amino acids are substituted withAlanine to form R4A/F6A-HN (this is named for the AGA triplet atlocations 4, 5, and 6 in the HN derivative. “HN17” is a truncated formof HN that includes 17 amino acids from Pro3 to Pro19.

A polypeptide having an amino acid sequence of:Pro-Xn1-(Cys/bXaa)-(Leu/Arg)-Xn2-Leu-Thr-(Gly/Ser)-Xn3-Pro (I) wherein“Cys/bXaa” indicates Cys or a basic amino acid; “(Leu/Arg)” indicatesLeu or Arg; “(Gly/Ser)” indicates Gly or Ser; and Xn1, Xn2, and Xn3independently indicate arbitrary amino acid sequences not more than 10residues in length, respectively;

A polypeptide having an amino acid sequence of:Pro-Xn1-(Cys/bXaa)-(Leu/Arg)-Xn2-Leu-Thr-(Gly/Ser)-Xn3-Pro (1).

Herein, “Cys/bXaa” indicates Cys or a basic amino acid; “(Leu/Arg)”indicates Leu or Arg; “(Gly/Ser)” indicates Gly or Ser; and Xn1, Xn2,and Xn3 independently indicate arbitrary amino acids not more than 10residues, respectively.

A polypeptide that has the amino acid sequence of:Pro-(Xaa)1-10-(Cys/bXaa)-(Leu/Arg)-(Xaa)1-10-Leu-Thr-(Gly/Ser)-(Xaa)1-10Pro(wherein Xaa indicates an arbitrary amino acid; “(Xaa)m-n” indicates mto n residues of arbitrary amino acids; “bXaa” indicates a basic aminoacid; “Cys/bXaa” indicates Cys or a basic amino acid; “(Leu/Arg)”indicates Leu or Arg; and “(Gly/Ser)” indicates Gly or Ser).

Basic amino acids refer to amino acids in which its R group (side chain)is positively charged at pH 7.0. Examples of natural basic amino acidsinclude Arg, Lys, and His.

The amino acid sequences of a polypeptide that has Arg, Lys, or His asthe basic amino acids can be represented, for example, as:Pro-Xn1-(Cys/Arg/Lys/His)-(Leu/Arg)-Xn2-Leu-Thr-(Gly/Ser)-Xn3-Pro(wherein “(Cys/Arg/Lys/His)” indicates Cys, Arg, Lys, or His;“(Leu/Arg)” indicates Leu or Arg; “(Gly/Ser)” indicates Gly or Ser; andXn1, Xn2, and Xn3 independently indicate arbitrary amino acids not morethan 10 residues, respectively). Herein, Arg and Lys can be the basicamino acid at this position.

Xn1, Xn2, and Xn3 are independently arbitrary amino acids of 2 to 6, 0to 4, and 2 to 6 residues, respectively (that is, Xn1=(Xaa)2-6,Xn2=(Xaa)0-4, and Xn3=(Xaa)2-6); or 3 to 5, 1 to 3, and 3 to 5 residues,respectively (that is, Xn1=(Xaa)3-5, Xn2=(Xaa)1-3, and Xn3=(Xaa)3-5); or4, 2, and 4 residues, respectively (that is, Xn1=(Xaa)4, Xn2=(Xaa)2, andXn3=(Xaa)4). Added amino acids of about 6 residues sometimes forms anα-helix and behaves like a single amino acid residue. A polypeptide ofthe present invention may be a polypeptide wherein arbitrary amino acidswith no more than 6 residues are added to all or any one of Xn1, Xn2,and Xn3 consisting of arbitrary amino acids of 4 residues, 2 residues,and 4 residues, respectively.

A sequence of Xn1 includes, for example, sequences consisting of(Arg/Ala)-(Gly/Ala)-(Phe/Ala)-(Ser/Ala), and sequences with conservativesubstitution thereof. Herein, for example, “Arg/Ala” indicates Arg orAla (“I” indicates that it is either one of the residues; the same isindicated throughout the description herein). Examples of such sequencesinclude Arg-Gly-Phe-Ser, Ala-Gly-Phe-Ser, Arg-Ala-Phe-Ser,Arg-Gly-Ala-Ser, Arg-Gly-Phe-Ala, and so on. Other examples includeArg-Gly-Ala-Ala, Arg-Ala-Phe-Ala, Arg-Ala-Ala-Ser, Arg-Ala-Ala-Ala,Ala-Gly-Phe-Ala, Ala-Gly-Ala-Ser, Ala-Gly-Ala-Ala, Ala-Ala-Phe-Ser,Ala-Ala-Phe-Ala, Ala-Ala-Ala-Ser, Ala-Ala-Ala-Ala, and such.

Conservative substitution can be exemplified by substitution within agroup of amino acids, corresponding to conservative substitution. On theother hand, the sequence of Xn2 includes, for example, sequencesconsisting of (Leu/Ala)-(Leu/Ala), and sequences with conservativesubstitution thereof. Such sequences include Leu-Leu, Ala-Leu, Leu-Ala,and such. Ala-Ala can be also exemplified as such sequences.Furthermore, the sequence of Xn3 includes, for example, sequencesconsisting of (Glu/Ala)-(Ile/Ala)-(Asp/Ala)-(Leu/Ala), and sequenceswith conservative substitution thereof. Such examples includeGlu-Ile-Asp-Leu, Ala-Ile-Asp-Leu, Glu-Ala-Asp-Leu, Glu-Ile-Ala-Leu,Glu-Ile-Asp-Ala, and so on. Other examples are Glu-Ile-Ala-Ala,Glu-Ala-Asp-Ala, Glu-Ala-Ala-Leu, Glu-Ala-Ala-Ala, Ala-Ile-Asp-Ala,Ala-Ile-Ala-Leu, Ala-Ile-Ala-Ala, Ala-Ala-Asp-Leu, Ala-Ala-Asp-Ala,Ala-Ala-Ala-Leu, Ala-Ala-Ala-Ala, and so on. The sequences of Xn1, Xn2,and Xn3 may be selected from arbitrary combinations.

Example 2 Experiment 5, the Effects of Humanin and its Analogs on MaleGerm Cell Apoptosis Induced by Chemotherapeutic Drugs

Advanced chemotherapies of cancer patients have increased survival andlife expectancy, and also increased expectations for a more favorableadverse effect profile. Of the long term adverse effects ofchemotherapies on cancer survivors, infertility has emerged as one ofthe chief complaints (Marcon L, et al., 2008; Meistrich ML., 2009; Lee SH, et al., 2013). Accordingly, the role of HN and HN analogs inpreventing male germ cell apoptosis induced by chemotherapeutic drugs inmouse models was explored. Doxorubicin (DOX) was chosen for the ex vivostudy as an anthracycline antibiotic that acts by intercalating DNA tosuppress proliferation and increase apoptosis and is active in vitro(Watring W G, et al., 1974). Cyclophosphamide (CP) was used in the invivo animal experiments because it requires liver cytochrome P450metabolism to become the activated form of the drug,4-hydroxy-cyclophosphamide, which circulates to cancer cells and damagesDNA leading to apoptosis (Gor P P, et al., 2010).

The effects of five HN analogs including HNG (HN-S14G, a potentagonist), HNG-F6A (no binding to IGFBP-3), HN-S7A (noself-dimerization), HN-C8P (no binding to BAX), and HN-L12A (a HNantagonist) on CP-induced male germ cell apoptosis in mice were studied.CP-induced germ cell apoptosis was inhibited by HN, HNG, HNG-F6A,HN-S7A, and HN-C8P (less effective), but not by HN-L12A. HN, HN-S7A, andHN-C8P restored CP-suppressed STAT3 phosphorylation. These resultssuggest that HN: 1) decreases DOX (ex vivo) and CP (in vivo) inducedmale germ cell apoptosis; 2) action is mediated by the membranereceptor/STAT3 with minor contribution by BAX-binding pathway; 3)self-dimerization or binding to IGFBP-3 may not be involved in HN'seffect in testis. This data suggests that HN is an important molecule inthe regulation of germ cell homeostasis after injury and agonisticanalogs may be developed for treating male infertility or protectionagainst chemotherapy side effects.

Materials and Methods Materials

HN peptide and the HN analogs were synthesized by CPC Scientific(Sunnyvale, Calif.). A description of the characteristics of each of theHN analogs is provided in Table 1. In in vivo experiments, HN and fiveHN analogs were studied by using a CP-induced male germ cell apoptosismouse model. These analogs include HNG (HN with a substitution of serine14 to glycine, HN-S14G) (Kunesová G, et al., 2008; Miao J, et al., 2008;Yamada M, et al., 2008), HNG-F6A (HNG with a substitution of alanine for6th phenylalanine, no binding to IGFBP-3)(Ikonen M, et al., 2003) HN-S7A(HN with a substitution of alanine for 7th serine, dimerizationdefective)(Yamagishi Y, et al., 2003), HN-C8P (HN with a substitution ofproline for 8th cysteine, no binding to BAX) (Hashimoto Y, et al., 2001;Guo B, et al., 2003; Yamagishi Y, et al., 2003), and HN-L12A (HN with asubstitution of alanine for 12th leucine, HN antagonist dimerizes withHN preventing HN binding to receptor)(Yamagishi Y, et al., 2003).

Mouse Seminiferous Tubule Ex Vivo Culture

A total of 15 mice were used for ex vivo studies. The animals weresacrificed and seminiferous tubules isolated from testes were cut intosmall segments under a dissecting microscope to identify light (earlystages I-IV and late stages XI-XII) of the seminiferous epithelium) anddark (middle stages VII-VIII) segments. Ten to twelve light segments (2mm in length) were placed per well on a six well plate with 2 ml serumfree medium of F-10 Ham nutrient mixture (Gibco, Life Technologies,Grand Island, N.Y.) following these treatments: control (Con, n=15 wheren is the number of times the treatment was repeated with segmentsobtained from different donor animals), heat (43° C., 15 min.; used as apositive control, n=10), HN 10 μg/mL (HN, n=9), doxorubicin 1 μM (Dox1,n=7), doxorubicin 1 μM+HN 10 μg/mL (Dox1+HN, n=7), doxorubicin 10 μM(Dox10, n=10), and doxorubicin 10 μM+HN 10 μg/mL (Dox10+HN, n=10). After12 hours of incubation at 34° C. with 5% CO2, the seminiferous tubulesfrom the seven treatment groups were used to make “squashed”seminiferous tubule preparations on a slide for TUNEL assay to detectgerm cell apoptosis (Erkkila K, et al., 1997). To quantify the rate ofgerm cell apoptosis, the squashed segments of seminiferous tubules wereexamined with an American Optical Microscope (Scientific Instruments,Buffalo, N.Y.) with a 40×objective and a 10×eyepiece lens. A square gridfitted within the eyepiece provided a reference area of 62,500 μm². TheTUNEL positive apoptotic germ cells within the frame of grid werecounted in 4 segments of seminiferous tubules in each group. Theincidence of germ cell apoptosis was expressed as the number ofapoptotic germ cells per mm².

Animals and In Vivo Experiments

Adult (12-week-old) male mice (C57BL/6J wild type, purchased fromJackson Laboratories, Bar Harbor, Me.) were used for animal experiments.All mice were housed in a standard animal facility under controlledtemperature (22° C.) and photoperiod of twelve hours of light and twelvehours of darkness with free access to food and water. Animal handlingand experimentation were in accordance with the recommendation ofAmerican Veterinary Medical Association and were approved by the animalcare and use review committee at the Los Angeles Biomedical ResearchInstitute at Harbor-University of California, Los Angeles (Harbor-UCLA)Medical Center.

For the HN analog experiments, male mice were divided into seven groups(n=4-5 per group) and received one of the following treatments andsacrificed after 24 hours: 1) vehicle (control); 2) a singleintra-peritoneal (IP) injection of HN peptide [HN, 40 mg/Kg body weight(BW)]; 3) a single IP injection of CP (CP, 200 mg/Kg BW); 4) IPinjection of CP and HN (CP+HN); 5) a single IP injection of each HNanalog (HNG 5 mg/Kg BW, HNG-F6A 5 mg/Kg BW, HN-S7A 40 mg/Kg BW, HN-C8P40 mg/Kg BW, or HN-L12A 40 mg/Kg BW); 6) IP injection of CP and each HNanalog (CP+HNG, HNG-F6A, HN-S7A, HN-C8P, or HN-L12A); and 7) IPinjection of CP+HN+HN analog (HN-S7A, HN-C8P, or HN-L12A; to assesswhether the analogs has enhancing or inhibitory effect on HN).

Tissue Preparation

To facilitate testicular fixation by using a whole-body perfusiontechnique, all animals were injected with heparin (130 IU/100 g BW, IP)15 min before a lethal injection of sodium pentobarbital (200 mg/kg BW,IP) (Lue Y H, et al., 1999). One testis was removed and weighed afterperfusion with saline. Portions of testicular parenchyma were snapfrozen in liquid nitrogen, and stored at −80 C for Western blotting. Theother testis was fixed by vascular perfusion with Bouin's solution, andprocessed with routine paraffin embedding for in situ detection ofapoptosis.

Western Blotting Analysis

Western blotting was performed as described previously (Jia Y, et al.,2009). In brief, proteins were denatured and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system(Invitrogen, Carlsbad, Calif.). After transferring, the Immuno-blot PVDFMembrane (Bio-Rad, Hercules, Calif.) was blocked for 1 h and then probedusing anti-STAT3 or anti-pSer727 STAT3 (Cell signaling Technology, Inc.,Beverly, Mass.) overnight at 4 C with constant shaking. After washing,membrane was then incubated with an anti-mouse (for STAT3 antibody,Santa Cruz Biotechnology, Santa Cruz, Calif.) or anti-rabbit (forpSer727 STAT3 antibody, Amersham Biosciences, Piscataway, N.J.) IgG-HRPsecondary antibody. All antibodies were diluted in blocking buffer. Forimmunodetection, membrane was incubated with enhanced chemiluminescencesolutions per the manufacturer's specifications (Amersham Biosciences,Piscataway, N.J.), and exposed to Hyperfilm ECL (Denville ScientificInc., Metuchen, N.J.).

Assessment of Apoptosis

Detection of apoptotic cells was performed in Bouin's′-fixed,paraffin-embedded testicular sections by the terminal deoxynucleotidyltransferase (TdT)-mediated deoxy-UTP nick end labeling (TUNEL) technique(Sinha Hikim A P, et al., 1997) using an ApopTag-peroxidase kit(Chemicon International, Inc., Temecula, Calif.). Enumeration of theSertoli cell nuclei with distinct nucleoli and apoptotic germ cellpopulation was quantified at stages I-IV (early stages), stages VII-VIII(middle stages) and stages XI-XII (late stages) of the seminiferousepithelial cycle using an Olympus BH-2 microscope (New Hyde Park, N.Y.).Stages were identified according to the criteria proposed by Russell etal for paraffin sections (Russell L, 1977). The rate of germ cellapoptosis (apoptotic index, AI) was expressed as the number of apoptoticgerm cells per Sertoli cells (Sinha Hikim A P, et al., 1997).

Statistical Analysis

Statistical analyses were performed using the SigmaStat 2.0 Program(Jandel Cooperation, San Rafael, Calif.). The Student-Newman-Keuls testafter one-way repeated measures ANOVA was used for statisticalsignificance. The seminiferous tubule culture experiments werereplicated 7-15 times and for animal experiments each group has 4-5mice. Differences were considered significant if P<0.05.

Results Effect of Doxorubicin on Germ Cell Apoptosis in SeminiferousTubule Cultures

In short term (12 hours) ex vivo seminiferous tubule cultures HN (10μg/mL) treatment alone had no significant effect on germ cell apoptosiswhen compared with control. As expected, heat exposure (43° C., 15 min.)serving as a positive control significantly increased the rate ofapoptosis compared to control and HN treated groups (p<0.05). HN (10μg/mL) prevented the heat induced germ cell apoptosis. Low dosedoxorubicin (1 μM) treatment increased apoptosis but did not reachstatistical difference because of the large variations compared withcontrol; addition of HN did not significantly decrease apoptosis (FIG.13). High dose doxorubicin (10 μM) treatment significantly increasedapoptosis compared to control (p<0.05); while HN (10 μg/mL)significantly reduced the number of apoptotic germ cells induced by highdose doxorubicin (p<0.05) (FIG. 13). In the squashed tubules, the germcells undergoing apoptosis were mainly pachytene spermatocytes and roundspermatids, no apoptotic spermatogonia were detected.

Effects of HN and HNG on CP-Induced Apoptosis in Testis

Synthetic HN or HNG peptide alone did not change the spontaneous germcell apoptosis. CP treatment increased germ cell apoptosis at bothearly+late stages (I-IV and XI-XII) (FIG. 2 upper panel, CP: 0.21±0.03,p<0.01 compared with control group, 0.04±0.01) and middle stages(VII-VIII) of seminiferous epithelial cycle in mice (bottom panel, CP:0.25±0.03; p<0.01 compared with control group, 0.01±0.01). CP-inducedgerm cell apoptosis was significantly (˜57%) inhibited by HNadministration (40 mg/kg BW) in early+late stages (stages I-IV andXI-XII) (FIG. 14A, CP+HN: 0.09±0.01, p<0.05 compared with CP treatedgroup), but not in middle stages (stages VII-VIII) (bottom panel, CP+HN:0.24±0.03; p>0.05 compared with CP treated group). Similarly, HNG at 5mg/kg BW was able to significantly inhibit (˜52%) the CP-induced germcell apoptosis in stages I-IV and XI-XII (FIG. 14A, upper panel, CP+HNG:0.10±0.01, p<0.05 compared with CP treated group) which was similar tothe effect of HN at 8-fold higher dose in early+late stages. HNG alsoprevented ˜36% of CP-induced germ cell apoptosis in middle stages(stages VII-VIII) (FIG. 14B, bottom panel, CP+HNG: 0.14±0.04 comparedwith CP group), although this effect did not reach statisticalsignificance (p>0.05). In this and all subsequent in vivo experimentsmainly pachytene spermatocytes and round spermatids but notspermatogonia underwent apoptosis

Effect of HNG-F6A, a HN Analog that does not Bind to IGFBP-3, on GermCell Apoptosis

In cell culture systems synthetic HNG-F6A peptide does not bind withIGFBP-3 (Ikonen M, et al., 2003). In present study, HNG-F6A alone didnot change spontaneous germ cell apoptosis. Like HNG, HNG-F6A at 5 mg/kgBW dose was able to inhibit (˜38%) the CP-induced germ cell apoptosis atstages I-IV and XI-XII (FIG. 15A, upper panel, CP+HNG-F6A: 0.13±0.02,p<0.05 compared with CP treated group: 0.21±0.01) which was similar tothe effect of HN at 8-fold higher dose (40 mg/kg BW) in early+latestages (I-IV and XI-XII). In addition, HNG-F6A also prevented ˜42% ofCP-induced germ cell apoptosis in middle stages (VII-VIII) (FIG. 15B,bottom panel, CP+HNG-F6A: 0.19±0.02 vs CP: 0.33±0.03, p<0.05).

Effects of HN Analogs that do not Dimerize (HN-S7A) or Bind to BAX(HN-C8P) on Male Germ Cell Apoptosis

Synthetic HN-S7A or HN-C8P peptide alone did not change the spontaneousgerm cell apoptosis. Similar to HN, CP-induced germ cell apoptosis inthe early and late stages (I-IV and XI-XII) was inhibited by same dose(40 mg/kg BW) of HN-S7A (FIG. 16A, CP+HN: 0.10±0.02 vs CP+HN-S7A:0.14±0.01, both p<0.05 when compared with CP group: 0.22±0.01). But inmiddle stages (VII-VIII), HN-S7A had better protective effect than HN(FIG. 16B, CP: 0.33±0.03, CP+HN: 0.32±0.03, CP+HN-S7A: 0.19±0.02, CP VsCP+HN-S7A p<0.05). Adding HN to HN-S7A+CP had no additional effect bothin early+late and middle stages (FIGS. 16A and 16B). HN-C8P on its ownhad no effect on germ cell apoptosis and showed less protection againstCP-induced apoptosis (FIG. 17A, HN-C8P ˜30% protection, vs ˜60% in HN,p<0.05), and no significant effect in middle (VII-VIII) stages which wassimilar to HN (FIG. 17B). Adding HN to HN-C8P+CP showed decreasedCP-induced apoptosis as CP+HN both in early+late (I-IV and XI-XII) andmiddle stages (VII-VIII) (FIGS. 17A and 17B).

Phosphorylation of Ser727 and Tyr705 leads to activation of STAT3.Immuno-blot analyses on testis homogenates showed that STAT3phosphorylation had no significant change with HN or either HN analogtreatment under basal conditions in mice (FIGS. 16C, 16D, 17C and 17D).CP treatment suppressed Ser727-phosphorylated STAT3 in testes (p<0.05,FIGS. 16C, 16D, 17C and 17D). HN, HN-S7A, and HN-C8P treatment restoredCP-induced Ser 727-phosphorylation of STAT3 (all p<0.05) (FIGS. 16C,16D, 17C and 17D). Adding HN to the combination of CP+HN-S7A orCP+HN-C8P did not further enhance STAT3 phosphorylation.

Effects of HN Antagonist, HN-L12A, on CP Induced Male Germ CellApoptosis

Synthetic HN analog HN-L12A alone did not change the spontaneous germcell apoptosis. CP-induced germ cell apoptosis was significantly (˜57%)inhibited by HN administration (40 mg/kg BW) in early+late stages(stages I-IV and XI-XII) (FIG. 18A, upper panel, AI of CP+HN: 0.13±0.01,p<0.05 compared with AI of CP group: 0.25±0.01), but not in middlestages (VII-VIII, FIG. 18B). HN-L12A was unable to rescue the CP-inducedgerm cell apoptosis either in middle (VII-VIII) or early+late stages(I-IV and XI-XII) (FIG. 18). Adding HN-L12A at the same dose as HN (40mg/kg BW) completely blocked the preventive effect of HN againstCP-induced germ cell apoptosis in early+late stages (I-IV and XI-XII)(FIG. 18A, CP: 0.25±0.01, CP+HN: 0.13±0.01, CP+HN+HN-L12A: 0.25±0.03).The blocking effect of HN-L12A on HN action was not shown in middlestages (VII-VIII, FIG. 18B) because HN and HN-L12A had no effect ofapoptosis in the middle stages.

Discussion

Infertility and sub-fertility are among the most common long-term sideeffects of chemotherapy. Studies in men and rodents show thatchemotherapy can result in decreased sperm count and reproductivecapacity (Marcon L, et al., 2008; Meistrich ML., 2009; Delbes G, et al.,2010; Dohle G R, 2010; Trost L W, et al., 2010). Increased survival withmodern chemotherapeutics has elevated the expectation of fertilitypreservation in cancer survivors (Lee S H, et al., 2013). CP and DOX aretwo drugs which are commonly used in the treatment of cancers, eitheralone or in combination with other chemotherapeutic agents. Thesemedications induce cancer cell apoptosis but also damage testicular germcells (Hashimoto Y, et al., 2004). By treating cultured mouseseminiferous tubule with DOX, it was demonstrated that HN significantlyreduced doxorubicin induced germ cell apoptosis ex vivo. CP has beenused in well characterized animal models to study the adverse effects onmale germ cells (Cai L, et al., 1997). HN significantly reducedCP-induced germ cell apoptosis in mice mainly in the early and latestages of the seminiferous tubule epithelium. Thus, HN preventschemotherapeutic agents-induced male germ cell apoptosis.

Recently, a putative trimetric receptor (with three components: CNTFRα,IL-27 receptor WSX-1, and gp130) was found on the neuronal cell membranethat mediates the neuro-protective activity of HN via the STAT3 pathway(Hashimoto Y, et al., 2005, Hashimoto Y, et al., 2009; Matsuoka M, etal., 2010). WSX-1 and gp130 (but not CNTFRα) and downstream activationof STAT3 is critical for HN-induced anti-apoptotic effects in male germcells. In this study, CP induced male germ cell apoptosis in vivo byreducing pSTAT3 in the testis and HN restored the CP-suppressed STAT3phosphorylation. This is consistent with the finding that HN reducedheat or GnRH-A-induced apoptosis by reversing the heat or GnRH-A inducedsuppression of STAT3 phosphorylation (Jia Y, et al., 2013). These datasupport that the STAT3 phosphorylation pathway is one of the mechanismsfor anti-apoptotic effect of HN in male germ cells. The ubiquitousexpression of STAT3 in the testis suggests that the action of HN mightnot only target germ cells.

In present study, the mechanisms of the protective effect of HN wereexplored by using different HN analogs. HN has 24 amino acids and oneamino acid change modifies its function (Yamada M, et al., 2008). Forexample, HNG (change of the 14th amino acid from serine to glycine,HN-S14G) is more potent than HN in preventing cell damage (Kunesová G,et al., 2008; Miao J, et al., 2008; Yamada M, et al., 2008). Using 1/8the dose of HN by injection, HNG showed similar cytoprotective effectagainst CP-induced germ cell apoptosis as HN, indicating that HNG ismore potent than HN in vivo. This finding is consistent with the notionthat HNG has greater protective effects on neuronal cells or tissuesthan HN. Double amino-acid substituted analog HNG-F6A does not bindIGFBP-3 (Ikonen M, et al., 2003). The present study showed HNG-F6A andHNG had similar protective effects against CP-induced germ cellapoptosis at equivalent doses, indicating that the cytoprotectiveeffects of HN and its more potent analogs on male germ cells isunrelated to its binding to IGFBP3. This is similar to analogousexperiments showing that HNG-F6A and HNG increases glucose stimulatedinsulin secretion to a similar extent in both isolated islets/cellculture and whole animals (Kuliawat R1, et al., 2013).

The sequence motif from Pro3 to Pro19 forms the core domain of the 24amino acid sequence of HN. Within the core domain, each amino acid,Pro3, Cys8, Leu9, Leu12, Thr13, Ser14, and Pro19, but not Ser7, havebeen found to be essential for the neuro-protective function of HN(Hashimoto Y, et al., 2001; Hashimoto Y, et al., 2001). HN-S7A is adimerization-defective mutant, i.e. HN-S7A does not self-dimerize anddoes not dimerize with HN. HN-S7A has no cytoprotective effect inneuronal cells in vitro indicating that the self-dimerization process isessential for HN's neuro-protective function culture (Yamagishi Y, etal., 2003). In one in vivo experiment, HN-S7A was effective inpreventing CP-induced germ cell apoptosis but did not affect the abilityof HN to protect CP-induced male germ cells apoptosis suggesting thatthe self-dimerization process may not be critical for HN'scytoprotective effect in testis in vivo. Thus the loss of thecytoprotective effect of HN-S7A in neuron cell culture experiments werenot replicated in vivo mouse experiments. The action of HN-S7A may bedifferent in vitro versus in vivo where interactions with HN-S7A mayenhance its activity. Three components (CNTFRα, WSX-1, and gp130) of ahumanin receptor were found on the neuronal cell membrane that mediatesthe neuro-protective activity of HN. Only WSX-1 and gp130 (but notCNTFRα) are critical for HN-induced anti-apoptotic effects in male germcells. The assembly of different subunits of membrane receptor of HN intestis may be one of the reasons that self-dimerization may not benecessary for HN's effect in testis as that in neuron. HN-S7A suppressedCP-induced male germ cell apoptosis both in early+late (stages I-IV andXI-XII) as well as middle stages (VII-VIII), while HN only suppressedCP-induced apoptosis in early+late but not in middle stages. Thissuggests that HN-S7A may have additional mechanism for protecting malegerm cells which require further identification. Western blot analysesshowed both HN and HN-S7A restored the CP-suppressed STAT3phosphorylation supporting that the mechanism of the cyto-protectiveeffects of HN and HN-S7A is mediated through the IL6-like trimericmembrane receptor.

Cysteine at position 8 is an amino acid essential for HN'sneuro-protective function (Hashimoto Y, et al., 2001; Guo B, et al.,2003; Hashimoto Y, et al., 2001; Sponne I, et al., 2004; Zhai D, et al.,2005; Zhai D, et al., 2005; Arakawa T, et al., 2008; Arakawa T1, et al.,2011). HN-C8P does not bind with BAX and shows no anti-apoptotic effectsin cell culture (Guo B, et al., 2003; Yamagishi Y, et al., 2003). Invivo HN-C8P partially prevented CP-induced germ cell apoptosis but wasless potent than HN. This finding indicates the intracellular binding toBAX may play a role, but not a major part, in protective effect of HN inCP-induced germ cell death which is consistent with previousobservations (Jia Y, et al., 2013). Western blot showed that HN-C8Prestored the CP-suppressed STAT3 phosphorylation which suggests but doesnot prove that the mechanism of HN-C8P's protective effect may bethrough membrane receptor.

The sequence motif from Leucine 9 to Leucine 12 plays a functional roleas a hydrophobic core of the HN peptide (Hashimoto Y, et al., 2001;Yamagishi Y, et al., 2003; Hashimoto Y, et al., 2001). Althoughreplacement of Leu12 with Alanine does not attenuate HN's secretoryactivity, HN-L12A lacks the ability to bind with membrane receptor andhas no anti-apoptotic effect in cell culture (Yamagishi Y, et al.,2003). HN-L12A dimerizes with HN prevents its binding to the receptorsacting as a HN antagonist. In the present study, HN-L12A did not preventCP-induced germ cell apoptosis but completely blocked the cytoprotectiveeffect of HN against CP-induced male germ cell apoptosis at similar doseof HN. These results indicated that membrane receptor binding ability isimportant for HN and HN analogs cytoprotective activity. Published datafrom other investigators showed that HN-S7A and HN-C8P had nocytoprotective effect in vitro (Hashimoto Y, et al., 2001; Yamagishi Y,et al., 2003; Hashimoto Y, et al., 2001), while these two HN analogspartially protected male germ cell from CP-induced apoptosis in vivo.The contrasting effects of HN analogs (such as HN-S7A and HN-C8P) invitro and in vivo suggest that HN and HN analogs may be modified in vivoor may act through different pathways or systems (e.g. IGF-1 pathway,(Yen K, et al., 2013)) instead of directly affecting the target cells ororgans. Arakawa et al studied the biological activity and structuralstabilities of HN analogs. Theses investigators also concluded thatdifferent biological activities of HN analogs could not be explainedonly by one simple factor (e.g. the structure stabilities) (Arakawa T1,et al., 2011). HN prevents male germ cell apoptosis induced by differentstress mainly by binding to the putative membrane receptor and STAT3pathway but HN also binds extra-cellularly to IGFBP-3 (Miao J, et al.,2008; Jia Y, et al., 2010) or intra-cellularly to BAX (Guo B, et al.,(2003); Zhai D, et al., 2005; Jia Y, et al., 2010), thus the mechanismof HN's anti-apoptotic effect in male germ cell is complex and may actthrough multiple pathways including in vivo modification of HN and itsanalogs. Understanding of the mechanisms of HN action may help to designspecific targets that may be used for drug development, e.g. agoniststhat protect male germ cells from stress-induced death (e.g. testicularhyperthermia, hormonal deprivation, or chemotherapy). In present study,the germ cells undergoing apoptosis were mainly pachytene spermatocytesand round spermatids. Apoptotic spermatogonia were not observed likelydue to the short duration of exposure to CP (12 hours ex vivo and 24hours in vivo). Male germ cells at different stages of the seminiferousepithelium have different mechanisms regulating the balance betweenapoptosis/survival homeostasis. Germ cells in early+late stages in acuteexperiments are more susceptible to heat stress than those middle stagegerm cells; while middle stages germ cell apoptosis could be induced bytestosterone deprivation (Lue Y H, et al., 1999; Lue Y, et al., 2006;Jia Y, et al., 2007). In the present study, HN showed preventive effectsagainst CP induced apoptosis in early+late but not in middle stages mostlikely because of the protective effect of intratesticular testosteronepresent in the middle stages. If the testis were examined after longerexposure to CP, it is expected that the early+late stage apoptosis willprogress to involve middle stage germ cells.

In summary, it was demonstrated that 1) HN significantly prevents malegerm cell apoptosis induced by chemotherapeutic agents both ex vivo andin vivo; 2) the anti-apoptotic effect of HN, HN-S7A, and HN-C8P onCP-induced male germ cell apoptosis is mainly mediated through the cellmembrane receptor and downstream STAT3 pathway; 3) lack of IGFBP-3binding ability does not neutralize HNG's cytoprotective effect intestis; 4) self-dimerization may not be necessary for HN preventingCP-induced germ cell apoptosis in vivo; 5) BAX-binding pathway may alsoplay a role in HN's protective effect; 6) HN-L12A is an antagonist to HNpreventing its cytoprotective effect in germ cell apoptosis in vivo. HNmay be an important molecule in the regulation of germ cell homeostasisand may have roles in male infertility and prevention ofchemotherapy-induced onco-infertility (Jia Y, et al., 2010).

Example 3 HNG Enhances Tumor Suppression by a Chemotherapeutic Agent CP

A mouse mammary cancer model was used which utilizes 4T1 cellstransfected with the luciferase gene. Growth and size of 4T1 mammarytumors were detected by bioluminescence imaging (IVIS Luminia II, PerkinElmer, Waltham, Mass.). Mice (n=5 to 7 per group) were inoculated with1×10⁶ 4 T1 cells into the right third mammary gland, and 7 days laterthey were left untreated or treated with HNG (5 mg/kg/day IP), CP (100mg/Kg single IP) or HNG+CP for 14 days. Bioluminescence imaging wasconducted at days 7 and 12. Animals were sacrificed and tumors wereremoved on day 14 after the start of treatment. Mammary tumors weresmaller in CP and CP+HNG groups compared to the untreated andHNG-treated animals (Data images not shown). Mammary tumors removed fromuntreated and HNG-treated mice were larger and heavier than the tumorsin the CP and CP+HNG group (p<0.001). Mean tumor weight was alsosignificantly lower in the HNG+CP compared to CP treated mice (p<0.012)(FIG. 19). This experiment showed that HNG enhanced the CP-inducedsuppression of 4T1 mouse mammary cancer growth.

Example 4 HNG Protects Ovaries from Chemotherapy—Induced Damage

A mouse mammary cancer model was used which utilizes 4T1 cellstransfected with the luciferase gene. Growth and size of 4T1 mammarytumors were detected by bioluminescence imaging (IVIS Luminia II, PerkinElmer, Waltham, Mass.). Mice (n=5 to 7 per group) were inoculated with1×10⁶ 4 T1 cells into the right third mammary gland, and 7 days laterthey were left untreated or treated with HNG (5 mg/kg/day IP), CP (100mg/Kg single IP) or HNG+CP for 14 days. Animals were sacrificed and bothovaries were removed from each mouse on day 14 after the start oftreatment. Serial 5-micrometer sections were made. Every 5th section wasexamined and the number of primordial, primary, and secondary folliclescounted (Kim S Y, et al., 2013; Bristol-Gould S K, et al., 2006; RotiRoti E C, et al., 2012). In addition, TUNEL staining for apoptotic cellsand oocytes was quantified (Perez G I, et al., 1997; Ben-Aharon I, etal., 2010). Representative sections of ovaries showed that control,non-treated tumor-bearing, and HNG treated mice have many primary,secondary, and antral follicles, whereas as CP treated ovaries have lesssecondary, but more atretic follicles. HNG treatment appeared to reversethe CP-induced decrease in follicles (FIG. 20).

Example 5 Humanin May be a Caloric Restriction Mimetic and can DecreasesIGF

Obesity increases the risks of developing breast cancer and recurrenceof the cancer (Biglia N, et al., 2013; Garrisi V M, et al., 2012;Kamineni A, et al., 2013; Simpson E R, et al., 2013). Caloricrestriction is associated with longevity, resistance to stress, andpostponement or attenuation of cancer growth, immunosenescence, andinflammation without permanent side effects across species (Bar-JosephH, et al., 2010; Desai V G, et al., 2013, Doroshow J H, et al., 1983;O'Brien P J, et al., 1997; Ottewell P D, et al., 2008). A long-termlow-fat diet in the Women's Health Initiative did not result in areduction in breast cancer risk. The reduction of fat intake after 6years was only 8.1%, well below the goal of fat intake of 20%,indicating that long-term adherence to diet restriction is difficult tomaintain (Chlebowski R T, et al., 1993; Prentice R L, et al., 2006). Thelife-prolonging effects of caloric restriction are most likely relatedto decreased IGF-1 levels (Ottewell P D, et al., 2008). Mice and mendeficient in GH, GH receptor, IGF-1, or IGFR are less susceptible tostress, are protected against age-related diseases including diabetesand cancer, and have longer life span (Ewans A, et al., 2006; Mayle A,et al., 2013; Rundberg Nilsson A, et al., 2013). Lowering IGF-1 levelscan result in inhibition of tumorigenesis (Schlueter A J, et al., 2001;Jimenez M, et al., 2005; Sun X, et al., 2001; Panosyan E H, et al.,2014).

After 72 hours of fasting, mice demonstrated lower glucose, IGF-1, andIGFBP-3 levels and increased IGFBP-1 levels (FIG. 21) with slight butnon-significant increases in GH and minimal increases in insulin (GoelS, et al., 2013). Mice bearing metastatic melanoma, exogenous HNG aloneor CP alone modestly suppressed IGF-1 but HNG+CP further suppressedIGF-1 levels (FIG. 22). Without being limited to theory, the suppressionof IGF-1 suggests that HNG may suppress tumor growth via the IGF-1signaling pathway (RAS/RAF/MAPK and PI3K/Akt/m-TOR) mimicking caloricrestriction pathways resulting in tumor growth suppression (FIG. 23).These preliminary data support a concept that HN is a caloricrestriction mimetic, and its cytotoxic effects in cancer may be mediatedby lowering IGF-1 without increasing GH and insulin.

Example 6 HNG Reduces Cyclophosphamide (CP)-Induced Germ Cell Apoptosis

Chemotherapeutic agents on mouse germ cells, a single dose ofcyclophosphamide (CP) at 200 mg/kg causes significant apoptosis of mousegerm cells after 24 hours. To examine the protective effects of HNG ongerm cells, adult mice were treated with vehicle (control); HNG (5mg/kg/BW, intraperitoneal injection IP); CP (200 mg/kg/BW, IP); andCP+HNG for 24 hours. CP treatment significantly increased germ cellapoptosis in all stages of spermatogenesis (P<0.001). HNG alone had noeffect, but HNG significantly decreased CP-induced apoptosis (p<0.05)(FIG. 24).

Example 7 The Mitochondrial Peptide Humanin (HN) is a Potent Inducer ofAutophagy

In several cell types including HEK293 (normal embryonic kidney cells),SH-SY5Y (neuroblastoma), and B16 (melanoma), HN induces autophagy. Theincreased level of LC3-II, a marker of autophagosome was examined byWestern blot and immunocytochemistry. The increase of autophagosomes andautolysosomes in HEK293 cells stably expressing mRFP-GFP-LC3, a dual-tagreporter of autophagy, was examined following humanin treatment. Inaddition, the signaling pathways activated by humanin using aphospho-antibody array in SH-SY5Y cells was investigated. This revealedseveral pathways known to regulate autophagy. Both activation andsuppression of autophagy are involved in cancer pathogenesis andtreatment, depending on the context.

This study was designed to determine the synergistic/additive effect ofhumanin on the efficacy of doxorubicin in B16 melanoma cells in cultureand more specifically, to reveal whether autophagy is involved in thiscombination strategy. Fluorescent microscopy and western blot of LC3-IIconfirmed that the potent humanin analog, HNG, increased autophagy inB16 cells. The combined treatment caused an additive effect on thecytotoxicity versus doxorubicin treatment alone. In addition, inhibitingautophagy by treating cells with autophagy inhibitors, including 3-MAand chloroquine, diminished the additive effect of humanin indoxorubicin treated B16 cells. Taken together, the current studysuggests that humanin has direct effects on tumor cells that involvesinduction of autophagy and thus humanin acts as a chemotherapeuticaugmenter and synergistically enhances doxorubicin's anticancer effects.Furthermore, the autophagy induction of humanin may be related to itsapparent effects of enhancing longevity observed in other systems.

Example 8 The Mitochondrial Peptide Analog HNG Protects AgainstCyclophosphamide-Induced Decrease in Sperm Output

The objective of this study was to investigate whether HNG hasprotective effect on sperm output after multiple doses of CP in mice.Thirty adult male mice (C57BL/6J) were randomized into 4 groups: 1) 5 ascontrol; 2) 5 received daily subcutaneously injection of HNG (10 mg/kgBW); 3) 10 were given 6 doses of CP (150 mg/kg BW) intraperitoneally at5-day intervals; 4) 10 received both HNG and CP. All mice were killed at28 days. Plasma HNG and IGF-1 levels were measured by ELISAs. The caudaepididymal sperm count was performed using a hemocytometer. Plasma HNGlevels increased significantly (p<0.001) in HNG treated (80.8±7.8ng/ml), and HNG+CP treated (64.7±1.8 ng/ml) mice compared to control(1.3±0.1 ng/ml), and CP treated mice (1.7±0.1 ng/ml). Compared tocontrol (413.7±44.9 ng/ml), plasma IGF-1 levels were significantly(p<0.001) suppressed by HNG (347.2±20.1ng/ml), CP (182.4±10.5 ng/ml),and further suppressed by CP+HNG treatment (148.8±8.1 ng/ml). Epididymalsperm counts were significantly elevated by HNG (1.7±0.2×10⁶/mg,p=0.04), and significantly suppressed by CP (0.5±0.1×10⁶/mg, p<0.001) ascompared to control (1.2±0.2×10⁶/mg)(FIG. 25). HNG+CP significantlyincreased sperm count (0.8±0.1×10⁶/mg, p=0.02) as compared to CP. It wasconcluded that HNG prevents CP-induced suppression of sperm output.These findings suggest that HNG is a promising adjuvant to chemotherapyby preventing onco-infertility.

Example 9 Adenosine Monophosphate-Activated Protein Kinase (AMPK)Pathway May be Involved in HNG Treatment in Pulmonary Mouse MetastaticMelanomas

Background: Humanin (HN) is a mitochondrial derived peptide withcytoprotective effects in normal cells challenged by various stressors.As shown herein, the potent synthetic HN analog (HNG, HN-S14G)attenuates male germ cell apoptosis induced by cyclophosphamide (CP)treatment in rodents while suppressing CP-induced tumor growth in amouse lung B16 melanoma metastasis model. HNG has been shown to activateseveral signal transduction pathways, including promoting AMPKsignaling. It was hypothesized that a possible mechanism for HNGenhanced chemotherapy-induced tumor suppression is by changing theenergy/nutrient availability by activating AMPK, and suppressing mTOR(mammalian target of rapamycin) dependent pathway.

Methods: Adult male mouse (n=5/group) were seeded with mouse B16melanoma cells (100,000 cells/mouse) via tail vein on day 1. One weekafter melanoma cell inoculation, mice were treated either with a singleintraperitoneal injection (IP) of CP (200 mg/kg), or daily IP injectionsof HNG (5 mg/kg), or both for 14 days. Mice were sacrificed at day 21and lungs were fixed in formaldehyde. Immunohistochemistry was performedon lung metastases with antibodies against phosphorylated-AMPK (p-AMPK),p-4E-BP1 (downstream effector of mTOR) and Ki-67 antigen (a marker ofcell proliferation).

Results: Compared to non-treated mice, HNG decreased number ofmetastatic lung tumors by ˜23%, CP by ˜50% and HNG+CP by ˜61%. Thepercent p-AMPK positive cells in metastatic lung melanoma treated withHNG alone (40.3±10.7, mean±SD) and HNG+CP (38.0±13.0) were significantlyincreased compared to CP alone (11.3±15.6, p<0.22). Expression of mTORdownstream effector for proliferation, p-4E-BP1, within the metastatictumors was very variable and not significantly different among groups.The mean Ki-67 index was significantly lower (p=0.0036) in CP treated(30.0±16.2) and CP+HNG groups (35.8±12.8), compared to untreated(62.0±14.8) and HNG groups (60.0±14.1SD). Co-administration of HNG withCP did not appear to further inhibit Ki-67 index.

Conclusion: HNG activates the key energy homeostasis regulator AMPKwithout affecting tumor mTOR signaling. The residual tumor cells thatsurvived chemotherapy may have adapted to energy deprivation. AMPKactivation may initially lead to mTORC1 (mTOR complex 1) inactivation,followed by mTORC2 mediated reactivation of mTORC1 resulting inincreased p-AMPK with no detectable change in p-4EBP1 expression in thetumor after CP±HNG treatment. Other pathways may also be involved toantagonize the inhibition of mTOR by AMPK in the tumor. This suggeststhat AMPK activation may be sufficient to reduce tumor growth withoutmTOR suppression. Importantly HN may have differential actions bymodulating the host via both AMPK and mTOR pathways mimicking reducednutrient availability in caloric restriction but increase AMPK signalingonly in the tumor.

Example 10 Humanin Analog (HNG) Prevents Male Germ Cell ApoptosisInduced by Temozolomide in Severe Combined Immuno-Deficiency (SCID) MiceBearing Medulloblastoma

Onco-infertility is a major concern of cancer survivors. In this study,it was investigated whether HNG will prevent chemotherapy (Temozolomide,TMZ, an alkylating agent used clinically to treat brain tumors) inducedmale germ cell apoptosis in SCID mice bearing subcutaneous implants ofhuman medulloblastoma. DAOY human pediatric medulloblastoma cells(1.0×10⁸ cells/animal) were injected subcutaneously into the right flankof 7-week old male SCID mice. After 3 weeks to allow tumor growth, eachgroup of mice (n=4 to 5/group) received one of the followingtreatments: 1) vehicle (control); 2) HNG intra-peritoneal (IP) injection(HNG, 5 mg/Kg BW/day, 3 days pretreatment+5 days treatment); 3) TMZ IPinjection (TMZ, 50 mg/Kg BW/day X 5 days); 4) TMZ and HNG IP injections(TMZ+HNG). Mice were sacrificed 24 hours after the last injection,subcutaneous tumors were dissected and weighed (FIG. 26) and male germcell apoptosis was assessed by TUNEL staining and quantified (ApoptosisIndex, AI, number of apoptotic germ cell/Sertoli cell, FIG. 27). TMZmarkedly suppressed tumor growth (FIG. 26); addition of HNG did notalter the marked suppression of tumor growth by TMZ. HNG alone had noeffect on male germ cell apoptosis, but significantly preventedTMZ-induced apoptosis in both middle stages (TMZ 0.058±0.006, TMZ+HNG0.023±0.003, p<0.05, FIG. 27A) and early+late stages (TMZ 0.116±0.009,TMZ+HNG 0.060±0.010, p<0.05, FIG. 27B). HNG ameliorated TMZ-induced germcell apoptosis without affecting the chemotherapeutic effect ontransplanted medulloblastoma tumor in an immunocompromised mouse model.This data indicates that HN and its analogs may be used as supportivetherapy to preserve fertility.

Example 11 Humanin Protects Against Chemotherapy-Induced Stage SpecificMale Germ Cell Apoptosis in Rats

Experiments were performed herein to examine whether HN has protectiveeffects on chemotherapy-induced male germ cell apoptosis and toinvestigate whether the protective effect of HN on germ cells requirethe presence of Leydig cells. The results show that HN decreased CPand/or EDS-induced germ cell apoptosis in a stage-specific fashion. HNacted directly on germ cells to protect against EDS induced apoptosisdespite depletion of Leydig cells and low intratesticular testosteronelevel in adult rats.

Materials and Methods Animals

Young adult (3-month-old) male Sprague Dawley rats used for the in vivoand in vitro studies were purchased from Charles River Laboratories(Wilmington, Mass., USA) and housed in a standard animal facility undercontrolled temperature (22° C.) and photoperiod of 12 h of light and 12h of darkness with free access to food and water. The animal useprotocol was reviewed and approved by the Institutional Animal Care andUse Committee of Los Angeles Biomedical Research Institute atHarbor-UCLA Medical Center.

Experiment a The Effects of HN on Chemotherapy-Induced Male Germ CellApoptosis with or without Pre-Treatment with EDS to Deplete Leydig Cellsin Rats

Thirty two young adult (60-day-old) male rats were randomly divided into8 groups with 4 rats per group. To examine the effects of HN onCP-induced male germ cell apoptosis, four groups of rats were treatedwith 1) vehicle (control); 2) a single intraperitoneal (IP) injection ofHN (40 mg/kg BW) (synthesized by CP scientific, Sunnyvale, Calif.); 3) asingle IP injection of CP (Cyclophosphamide Monohydrate, 70 mg/kg BW,Sigma, St. Louis, Mo.); or 4) CP+HN to rats. To determine whether Leydigcells were involved in the cytoprotective action of HN on germ cellapoptosis, 4 groups of rats were pre-treated with IP injection of ethanedimethanesulfonate (EDS, 80 mg/kg) to eliminate Leydig cells. EDS was agift from M. Meistrich, PhD, MD Anderson Cancer Center. EDS wassynthesized by the M.D. Anderson Translational Chemistry Core Facilityunder the direction of Dr. William Bornmann. After 3 days, whendepletion of Leydig cells occurred (Morris et al, 1997), a single IPinjection of vehicle (control); HN; CP; or HN+CP to 4 groups of EDSpre-treated rats was administered. Twelve hours after treatment, allrats were injected with heparin (130 IU/100 g BW, i.p.) 15 min beforebeing sacrificed by a lethal injection of sodium pentobarbital (200mg/kg BW i.p.) to facilitate testicular perfusion using a whole-bodyperfusion technique (Lue, et al., 2010). Body weight was recorded atautopsy and blood samples were collected from the right ventricle ofeach animal immediately after death, and plasma was separated and storedat −20 C for subsequent T measurement. One testis from each animal wassnap-frozen in liquid nitrogen. The contralateral testis was then fixedby vascular perfusion with Bouin's solution for 40 min. preceded by abrief saline wash. Testes were removed and placed into the same fixativeovernight. One slice from the middle region of Bouin's fixed testis wasprocessed for routine paraffin embedding for histological evaluation andterminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)assay (Lue, et al., 2010).

Experiment B The Effects of HN on Cultured Leydig Cells Treated withKetoconazole

Leydig cells were isolated based on a previously described procedure(Gao, et al., 1996; Klinefelter, et al., 1987; Salva, et al., 2001).Briefly, the testes were decapsulated, and then dissociated withcollagenase, dispase, DNase and shaking for 15 min at 34° C. Theseminiferous tubules were removed by filtration through 40 μm nylon mesh(BD Falcon, Franklin Lakes, N.J.). Leydig cells were then harvestedafter centrifugation and purified using a Percoll gradient (GEHealthcare, Uppsala, Sweden) and centrifugation (60 minutes at 20,000 gat 4° C.). Enriched Leydig cells were harvested at densities between1.065 (red) and 1.075 (blue) g/cm³ from the percoll gradient. TheseLeydig cells were washed by diluting the percoll and excluding residualgerm cells and other cells using a BSA density gradient withcentrifugation. The purity of the Leydig cells was >90%, as determinedby histochemical staining for 3β-hydroxysteroid dehydrogenase. The cellviability, as assessed by trypan blue exclusion, was greater than 90%.In all the in vitro experiments, 2×10⁵ purified Leydig cells were addedto each well of the 6-well plates in 2 ml Leydig cell culture media(Dulbecco's Modified Eagles Medium (DMEM)-Ham's nutrient mixture F-12(Life Technologies, Grand Island) containing penicillin and streptomycin(Invitrogen Life Technologies, Inc., Paisley, UK). Eight replicateexperiments were performed where Leydig cells were incubatedrespectively with vehicle (control), HN (10 mcg/ml), KTZ (10 mcg/ml,Sigma Aldrich, St. Louis, Mo.), or KTZ+HN at 34° C. for 4 hours. Aftertreatment, the culture medium from each well was collected and stored at−20° C. for testosterone measurement.

Immunohistochemistry for Localization of HN in Testes

Endogenous HN localization in rat testes was detected byimmunohistochemistry using rat humanin (rattin) specific antibody. Inbrief, after de-paraffinization and rehydration, testicular sectionswere first incubated with a rabbit polyclonal anti rat-humanin (rattin)antibody (Abcam, Cambridge, Mass.) at a concentration of 10 mcg/ml at 4°C. overnight and then followed by Alexa Fluor-594 conjugated anti-rabbitsecondary antibody (Invitrogen, Life Technologies, Grand Island, N.Y.)at a concentration of 20 mcg/ml for 1 hour at room temperature. Fornegative controls, sections were treated only with secondary antibody.Slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) andreviewed with a Zeiss Axioskop 40 fluorescent microscope.

TUNEL Assay for Assessment of Apoptotic Cells in Testicular Sections

The in situ detection of cells with DNA strand breaks by terminaldeoxynucleotidyl transferase dUTP nick end labeling (TUNEL) wasperformed in paraffin-embedded testicular sections by using ApopTagPeroxidase in Situ Apoptosis Detection Kit (Millipore, Billerica, Mass.)as described earlier (Lue, et al., 2000). Negative and positive controlswere carried out in every assay. For negative controls, tissue sectionswere processed in an identical manner, except that the terminaldeoxynucleotidyl transferase enzyme was substituted with the same volumeof PBS. The apoptotic germ cell population was enumerated out bycounting TUNEL positive germ cells using the Axioskop 40 microscope(Zeiss, Thornwood, N.Y.). The rate of germ cell apoptosis (ApoptoticIndex, AI) was expressed as the percentage (%) of the number of thecross sections of seminiferous tubules containing TUNEL positiveapoptotic germ cells/number of cross sections of all seminiferoustubules at early (I-VI) and late (IX-XIV), and middle (VII-VIII) stageson the slide. In addition, the different types of apoptotic germ cellswere assessed using the same testicular sections.

Testosterone Measurement

Testosterone concentrations in plasma, testes, and culture medium weremeasured by a previously described radioimmunoassay (Lue, et al., 2010)with a minimal detection limit of 0.1 ng/ml. The intra- and inter-assaycoefficients of variations were 8 and 11%, respectively.

Statistical Analysis

Statistical analyses were performed using SAS 9.3 (SAS Institute, Carey,N.C.). The in vivo and in vitro data were analyzed by unpaired Student'st-test after confirmation by Shapiro-Wilk test to reject statisticallysignificant non-normality. For the in vivo experiments it was firstassessed whether or not CP, EDS or both increased the AI using theunpaired student t-test by comparing CP vs control, EDS vs control andEDS+CP vs control, respectively. Contrasts of a priori interest werethen examined which were focused on the effects of HN: CP vs CP+HN; EDSvs EDS+HN; and EDS+CP vs EDS+CP+HN. Statistical comparison of AI inthese groups of interest were shown in the figures. For the in vitroanalyses, the experiment was designed to determine if HN could salvagetestosterone production (treated with KTZ) from Leydig cells in vitro.Accordingly, it was first assessed whether or not KTZ reducedtestosterone by unpaired student t-test by comparing KTZ vs control. Itwas next assessed, by unpaired student t-test, whether HN preventingthis occurring by comparing KTZ vs KTZ+HN. Statistical significance wasconstrued when <0.05. All tests were two-sided.

Results Rat HN Protein (Rattin) is Expressed in Leydig Cells and GermCells in Rat Testis

Using immunohistochemistry and a specific antibody against rat HN (alsonamed rattin in rat), it was demonstrated that rat HN was expressed inLeydig cells, spermatocytes and round spermatids. HN appearedpredominantly localized in the cytoplasm of Leydig cells, and moderatelyin cytoplasm of spermatocytes and round spermatids in adult rat testes.

EDS Eliminated Leydig Cells and Suppressed Testosterone Production

Histology of testes obtained from rats treated with EDS for three daysshowed the expected depletion of Leydig cells in the interstitial spacescompared to control animals. HN administered 3 days after EDSpre-treatment did not reverse the EDS-induced Leydig cell loss. In allgroups treated with EDS, intratesticular (FIG. 28A) and serumtestosterone levels (FIG. 28B) were decreased to very low levels (bothp<0.0005) compared to those without EDS pre-treatment. CP or HN had nosignificant effects (p>0.05 in all comparisons) on serum andintratesticular testosterone levels with or without EDS pre-treatment(FIG. 28).

HN Attenuates CP-Induced Germ Cell Apoptosis in Early (I-VI) and Late(IX-XIV) Stages of the Seminiferous Epithelial Cycle

CP was used to induce germ cell apoptosis and examined thecytoprotective efficacy of HN on germ cells against apoptosis in adultrats with or without EDS pre-treatment. In both FIG. 29A and FIG. 29B,the light bars represent animals not pre-treated with EDS where Leydigcells were present in the interstitial space, and the dark barsrepresent EDS pre-treated animals with testes depleted of Leydig cells.FIG. 29A presents germ cell apoptosis index (AI) obtained in early(I-VI) and late (IX-XIV) stages, and FIG. 29B middle (VII-VIII) stagesof the seminiferous epithelium cycle.

In these short term experiments, CP significantly increased germ cellapoptosis at early (I-VI) and late (IX-XIV) stages of the seminiferousepithelial cycle compared to control (p=0.0019) (FIG. 29A. light bars).At these early and late stages, CP treatment mainly induced apoptosis ofspermatocytes, round spermatid, and differentiated spermatogonia. HNco-treatment significantly reduced CP-induced apoptosis at early andlate stages (p=0.021, FIG. 29A, light bars) of seminiferous epithelialcycle in adult rats. Because CP treatment at this dose level for such ashort duration did not increase apoptosis in the middle (VII-VIII)stages of the seminiferous epithelial cycle (p=0.328), HN had no effecton CP treated testis (p=0.427)(FIG. 29B, light bars).

HN Attenuates EDS Induced Germ Cell Apoptosis in Middle Stages(VII-VIII) of the Seminiferous Epithelial Cycle

Pre-treatment with EDS had significant acute cytotoxic effects on germcells (p=0.0014) when compared to control in the androgen sensitivemiddle (VII-VIII) stages (FIG. 29B, dark bars). EDS pre-treatment mainlyinduced apoptosis of preleptotene and pachytene spermatocytes, roundspermatids, and elongated spermatids at the middle stages. The EDSeffect may be a direct cytotoxic effect or mediated by the extremely lowintratesticular testosterone levels and the depletion of Leydig cells.HN administered 3 days after treatment by EDS significantly reduced theEDS-induced apoptosis of germ cells (p=0.018). Addition of CP treatmentin EDS pre-treated rats increased germ cell apoptosis (p=0.0011) ascompared to control, but did not additively increased germ cellapoptosis caused by EDS treatment at middle stages (p=0.248). Theincrease in apoptosis in CP+EDS group was reduced by concomitant HNtreatment (p=0.04) at the middle-stages (FIG. 29B dark bars).

EDS pre-treatment did not significantly increase germ cell apoptosis atthe early (1-VI) and late (IX-XIV) stages compared to controls (p=0.169)and addition of HN did not significantly reduced germ cell apoptosis(p=0.251) (FIG. 29A, dark bars). Similarly in these early and latestages, CP also did not significantly increased germ cell apoptosiscompared to control (p=0.617) and HN did not change the CP effects inEDS pre-treated animals where Leydig cells were absent (p=0.173)(FIG.29A dark bars). However the comparison of the CP effect on germ cellapoptosis in rats with or without EDS pre-treatment (CP in FIG. 29A,light versus dark bars) showed that when Leydig cells were depleted andintratesticular testosterone was very low, CP induced germ cellapoptosis was significantly lower in EDS pre-treated rats (p=0.0023).

HN has No Effect on KTZ-Induced Suppression of Testosterone (T) ofLeydig Cells in Vitro.

KTZ, compared to control, lowered the testosterone levels significantly(p=0.038, FIG. 30) within in 4 hours. HN treatment, compared withcontrol, did not salvage the KTZ induced decrease in testosteroneproduction by the Leydig cells in vitro.

Discussion

Testicular stressors that induce germ cell apoptosis include agents thatlower intratesticular testosterone, testicular hyperthermia, and agentsthat can cause direct germ cell damage (e.g. alkylating agents such asCP).

In this study using a rat humanin (rattin) specific antibody, it wasobserved that rat HN was expressed in stress-sensitive primaryspermatocytes and round spermatids.

The chemotherapeutic agent CP was used to induce germ cell apoptosis. Inthese studies, it was demonstrated that HN was able to differentiallyprotect against CP-induced stage-specific (early and late) germ cellapoptosis in rats.

When Leydig cells were eliminated by EDS, CP failed to increase germcell apoptosis in early and late stages which were not sensitive toandrogens. This data suggests that Leydig cells may be involved inCP-induced germ cell apoptosis and absence of Leydig cells wasprotective for germ cells. In prior studies, GnRH antagonist plus theanti-androgen flutamide treatment suppressed testosterone levels andaction in rats and protected spermatogonial stem cell damage induced byCP or irradiation and restored spermatogenesis and fertility (Meistrich,et al., 1995; Shetty & Meistrich, 2005). However, when the sameprinciple was translated to non-human primates, suppression oftestosterone by GnRH antagonist did not restore spermatogenesis inducedby irradiation indicating species differences in testicular responses toGnRH antagonist and irradiation (Boekelheide, et al., 2005; Shetty &Meistrich, 2005). The observation that CP-induced apoptosis of germcells requires Leydig cells and testosterone does not distract from thepoint that HN protects against germ cell apoptosis in the absence ofLeydig cells and despite the fact that intratesticular testosteronelevels are very low.

To show that the cytoprotective effect of HN againstchemotherapy-induced apoptosis of male germ cells was a direct effect ongerm cells required evidence that the cytoprotective effects of HN didnot require Leydig cell and its products. EDS is a cytotoxic agent thateffectively eliminates Leydig cell by activating caspase-3 inducingLeydig cell apoptosis (Morris, et al., 1997; Rommerts, et al., 2004;Rommerts, et al., 1988; Sprando, et al., 1990). By selectivelyeliminating Leydig cells with EDS in the present study, it wasdemonstrated that EDS induced germ cell apoptosis in short termtreatment mainly at the androgen sensitive middle stages of theseminiferous epithelium but not at non-androgen sensitive early and latestages. This suggests that EDS induces apoptosis by reducingintratesticular testosterone to very low levels. Administration ofexogenous HN protected germ cells at the middle stages from apoptosis inresponse to EDS despite the absence of Leydig cells and testosterone.Although effects of HN on serum and intratesticular testosterone levelin vivo were not observed (FIG. 28), the possibility that this was dueto a large variation of serum and intratesticular T levels or a smallnumber of animals examined could not be ruled out. It was also observedthat CP failed to enhance EDS induced germ cell apoptosis in the middlestages. This might be due to the following: 1) absence of testosteroneprotects germ cell from CP action (Meistrich, et al., 1995; Shetty &Meistrich, 2005); 2) CP does not affect germ cell apoptosis in themiddle stages after 12 hours treatment; and 3) the very potent cytotoxiceffect of pre-treatment of EDS may have obliterated the small effect ifany of CP in the middle stages. When there was no increase in CP-inducedapoptosis in middle stages or EDS-induced apoptosis in early or latestages, HN has no effect on promoting germ cell survival.

Data herein demonstrates that HN protects against CP-induced germ cellapoptosis at early and late stages. HN reduced EDS-induced germ cellapoptosis at middle stages despite depletion of Leydig cells by EDS.Thus, the data herein provides strong supportive evidence that HN hasdirect cytoprotective action on male germ cells after chemotherapy in astage-specific fashion. It should be emphasized that the stagespecificity of the cytoprotective effects of humanin are evident inshort term experiments. It was anticipated that chronic treatment withcytotoxic agent will spread from the early and late stages where theybegin, to all stages of the seminiferous epithelium. Once apoptosisoccurs in one stage(s), with time and with continued treatment, thespermatogenic cycle will be arrested. If spermatogonia are damaged bythe cytotoxic agent, the spermatogenic cycle may not recover leading toonco-infertility.

Endogenous HN is expressed in both immature and adult Leydig cells(Colon, et al., 2006); the latter is confirmed in the current study. Toinvestigate the direct effects of HN on testosterone production fromLeydig cells, in vitro experiments were performed to investigatesynthetic HN's action on cultured adult Leydig cells treated withketoconazole (KTZ). KTZ is known to reduce testosterone levels by actingat multiple steps in testosterone synthesis. When HN was added to Leydigcells in vitro, synthetic HN at a dose examined was not able to preventthe decreased testosterone production of Leydig cells induced bytreatment with KTZ.

In summary, it was demonstrated that with short term treatment: 1) HNsignificantly reduced CP and EDS induced germ cell apoptosis atdifferent stages of the seminiferous epithelium; 2) HN protectedEDS-induced germ cell apoptosis in middle stages of the seminiferouscycle despite depletion of Leydig cells and lower intratesticulartestosterone levels in adult rats.

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The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. In caseof conflict, the specification, including definitions will control.Citation of the above patents, patent applications, publications anddocuments is not an admission that any of the foregoing is pertinentprior art, nor does it constitute any admission as to the contents ordate of these publications or documents.

Modifications may be made to the foregoing without departing from thebasic aspects of the technology. Although the technology has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments specifically disclosed in thisapplication, yet these modifications and improvements are within thescope and spirit of the technology.

The technology illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising,” “consisting essentially of,” and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and use of such terms and expressions do not exclude anyequivalents of the features shown and described or portions thereof, andvarious modifications are possible within the scope of the technologyclaimed.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

As used herein, the singular forms “a”, “and,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, the term“a” or “an” can refer to one of or a plurality of the elements itmodifies (e.g., “a reagent” can mean one or more reagents) unless it iscontextually clear either one of the elements or more than one of theelements is described.

The term “about” as used herein refers to a value within 10% of theunderlying parameter (i.e., plus or minus 10%), and use of the term“about” at the beginning of a string of values modifies each of thevalues (i.e., “about 1, 2 and 3” refers to about 1, about 2 and about3). For example, a weight of “about 100 grams” can include weightsbetween 90 grams and 110 grams. Further, when a listing of values isdescribed herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) thelisting includes all intermediate and fractional values thereof (e.g.,54%, 85.4%).

It is understood that although the present technology has beenspecifically disclosed by representative embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and such modificationsand variations are considered within the scope of this technology.

Certain embodiments of the technology are set forth in the claim(s) thatfollow(s).

What is claimed is:
 1. A method of protecting germ cells in a subjectfrom cell death, wherein germ cell death is stimulated by achemotherapeutic agent, comprising administering to a subject treatedwith a chemotherapeutic agent an amount of humanin or a humanin analogsufficient to protect germ cells in the subject from cell death.
 2. Amethod of inhibiting decreased or reduced fertility in a subject causedby treatment with a chemotherapeutic agent, comprising administering toa subject treated with a chemotherapeutic agent an amount of humanin ora humanin analog sufficient to inhibit decreased or reduced fertility inthe subject caused by treatment with a chemotherapeutic agent.
 3. Themethod of claim 1 or 2, wherein the subject has a hyperproliferativedisorder.
 4. The method of claim 1 or 2, wherein the subject has ametastatic or non-metastatic neoplasia, tumor, cancer or malignancy. 5.The method of claim 1 or 2, wherein the humanin or a humanin analog doesnot substantially reduce, decrease, suppress or inhibit efficacy oractivity of the chemotherapeutic agent.
 6. The method of claim 5,wherein the efficacy or activity of the chemotherapeutic agent comprisespartial or complete destruction of a hyperproliferating cell, or aneoplastic, tumor, cancer or malignant cell mass, volume, size ornumbers of cells; stimulating, inducing or increasing hyperproliferatingcell or neoplastic, tumor, cancer or malignant cell necrosis, lysis orapoptosis; reduces hyperproliferating cell or neoplasia, tumor, canceror malignancy volume size or cell mass; inhibits or prevents progressionor an increase in hyperproliferating cell or neoplasia, tumor, cancer ormalignancy volume, mass, size or cell numbers, reduces neoplasia, tumor,cancer or malignancy metastasis volume, size or cell mass; or prolongslifespan.
 7. The method of claim 1 or 2, wherein the humanin comprisesthe sequence: MAPRGFSCLLLLTSEIDLPVKRRA.
 8. The method of claim 1 or 2,wherein the humanin analog comprises the sequence:MAPRGFSCLLLLTGEIDLPVKRRA (HN-S14G), or any sequence set forth in Tables1-4.
 9. The method of claim 1 or 2, wherein the chemotherapeutic agentcomprises an alkylating agent, an anthracycline, an anti-metabolite,plant extract, plant alkaloid, nitrosourea, hormone, nucleoside ornucleotide analog.
 10. The method of claim 1 or 2, wherein thechemotherapeutic agent comprises a DNA intercalating agent or an agentthat attaches or bonds to DNA.
 11. The method of claim 1 or 2, whereinthe chemotherapeutic agent comprises cyclophosphamide, doxorubicin,azathioprine, cyclosporin A, prednisolone, melphalan, chlorambucil,mechlorethamine, busulphan, methotrexate, 6-mercaptopurine, thioguanine,5-fluorouracil, cytosine arabinoside, 5-azacytidine (5-AZC) and5-azacytidine related compounds, bleomycin, actinomycin D, mithramycin,mitomycin C, carmustine, lomustine, semustine, streptozotocin,hydroxyurea, cisplatin, carboplatin, oxiplatin, mitotane, procarbazine,dacarbazine, a taxane, vinblastine, vincristine, dibromomannitol,gemcitabine, or pemetrexed.
 12. The method of claim 4, wherein theneoplasia, tumor, cancer or malignancy is metastatic, non-metastatic orbenign.
 13. The method of claim 4, wherein the neoplasia, tumor, canceror malignancy comprises a solid cellular mass.
 14. The method of claim4, wherein the neoplasia, tumor, cancer or malignancy compriseshematopoietic cells.
 15. The method of claim 4, wherein the neoplasia,tumor, cancer or malignancy comprises a carcinoma, sarcoma, lymphoma,leukemia, adenoma, adenocarcinoma, melanoma, glioma, glioblastoma,medulloblastoma, meningioma, neuroblastoma, retinoblastoma, astrocytoma,oligodendrocytoma, mesothelioma, reticuloendothelial, lymphatic orhaematopoietic neoplasia, tumor, cancer or malignancy.
 16. The method ofclaim 15, wherein the sarcoma comprises a lymphosarcoma, liposarcoma,osteosarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma orfibrosarcoma.
 17. The method of claim 15, wherein the haematopoieticneoplasia, tumor, cancer or malignancy comprises a myeloma, lymphoma orleukemia.
 18. The method of claim 4, wherein the neoplasia, tumor,cancer or malignancy comprises a metastatic melanoma.
 19. The method ofclaim 4, wherein the neoplasia, tumor, cancer or malignancy comprises alung, thyroid, head or neck, nasopharynx, throat, nose or sinuses,brain, spine, breast, adrenal gland, pituitary gland, thyroid, lymph,gastrointestinal (mouth, esophagus, stomach, duodenum, ileum, jejunum(small intestine), colon, rectum), genito-urinary tract (uterus, ovary,cervix, endometrial, bladder, testicle, penis, prostate), kidney,pancreas, liver, bone, bone marrow, lymph, blood, muscle, or skin, lung,biliary tract, or hematologic neoplasia, tumor, or cancer.
 20. Themethod of claim 1 or 2, further comprising administering a second, thirdor fourth chemotherapeutic agent.
 21. The method of claim 1 or 2,wherein the humanin or humanin analog is administered prior to,substantially contemporaneously with or following administration of thechemotherapeutic agent.
 22. The method of claim 1 or 2, wherein thehumanin or humanin analog is administered in combination with thechemotherapeutic agent.
 23. The method of claim 1 or 2, wherein thesubject has undergone surgical resection, chemotherapy, immunotherapy,ionizing or chemical radiotherapy, local or regional thermal(hyperthermia) therapy, or vaccination.
 24. The method of claim 1 or 2,wherein the subject is or is not a candidate for surgical resection,chemotherapy, immunotherapy, ionizing or chemical radiotherapy, local orregional thermal (hyperthermia) therapy, or vaccination.
 25. The methodof any of claims 1 to 24, wherein the subject is a mammal.
 26. Themethod of any of claims 1 to 24, wherein the subject is a human.
 27. Themethod of any of claims 1 to 24, wherein the subject is a human male.28. The method of any of claims 1 to 24, wherein the subject is a humanfemale.
 29. The method of any of claims 1 to 24, wherein the cell deathis apoptosis.